Production of engineered t-cells by sleeping beauty transposon coupled with methotrexate selection

ABSTRACT

Aspects of the invention described herein include methods of treating, inhibiting, ameliorating and/or eliminating a virus or cancer cells in a subject utilizing genetically engineered human T-cells having receptors for a molecule presented by the virus or the cancer cells, wherein the genetically engineered T cells are isolated utilizing a two-stage MTX selection that employs increasing concentrations of MTX.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of priority to U.S.Provisional Patent Application No. 62/058,973, filed Oct. 2, 2014, U.S.Provisional Patent Application No. 61/977,751, filed Apr. 10, 2014, U.S.Provisional Patent Application No. 61/986,479, filed Apr. 30, 2014, U.S.Provisional Patent Application No. 62/089,730 filed Dec. 9, 2014, U.S.Provisional Patent Application No. 62/090,845, filed Dec. 11, 2014, andU.S. Provisional Patent Application No. 62/088,363, filed Dec. 5, 2014.The entire disclosures of the aforementioned applications are herebyexpressly incorporated by reference in their entireties.

REFERENCE TO SEQUENCE LISTING, TABLE, OR COMPUTER PROGRAM LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledSCRI.077PR.TXT, created Mar. 20, 2015, which is 4 kb in size. Theinformation is the electronic format of the Sequence Listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

Aspects of the invention described herein include methods of treating,inhibiting, ameliorating and/or eliminating a virus or cancer cells in asubject utilizing genetically engineered human T-cells having receptorsfor a molecule presented by the virus or the cancer cells.

BACKGROUND OF THE INVENTION

Engineered human T-cells are a promising therapeutic route for cancerimmunotherapy and viral therapy. T-cells expressing chimeric antigenreceptors combined with additional genes to enhance T-cellproliferation, survival, or tumor homing can further improve efficacybut require multiple stable gene transfer events. Accordingly, methodsare needed to increase production efficiency for multiplexed engineeredcells. Efficient, stable transduction of T-cells can be achieved using aSleeping Beauty transposon system in minicircles that are introduced bynucleofection. Rapid selection of transduced cells with methotrexate(MTX) for cells expressing a mutant dihydrofolate reductase (DHFRdm)resistant to metabolic inhibition can also be achieved.

SUMMARY OF THE INVENTION

Described herein are approaches for the preferential amplification of Tcells expressing multiple transgenes, preferably encoding receptors orchimeric receptors specific for a molecule presented by a virus or acancer cell. In some alternatives, selection pressure on transformed Tcells is applied in a two-stage MTX selection utilizing increasingconcentrations of MTX.

In one alternative, a gene delivery polynucleotide for stable insertionof a nucleic acid into an oligonucleotide is provided, wherein thenucleic acid for insertion is flanked by inverted terminal repeat genesequences in the gene delivery polynucleotide and wherein the genedelivery polynucleotide is selectable is provided, wherein the genedelivery polynucleotide comprises a first sequence, wherein the firstsequence comprises a first inverted terminal repeat gene sequence, asecond sequence, wherein the second sequence comprises a second invertedterminal repeat gene sequence, a third sequence, wherein the thirdsequence comprises a promoter region sequence, a fourth sequence,wherein the fourth sequence comprises at least one gene encodes aprotein or encodes a sequence for mRNA transcription, and wherein thefourth sequence is optimized, a fifth sequence, wherein the fifthsequence comprises at least one selectable marker cassette encoding adouble mutant of dihydrofolate reductase, wherein the double mutant ofdihydrofolate reductase has a 15,000 fold or about 15,000 fold reducedaffinity for methotrexate, wherein the methotrexate can be used toselect for cells transduced with the gene delivery polynucleotide toenhance the ratio of cells expressing the at least one gene and whereinthe fifth sequence is optimized, a sixth sequence, wherein the sixthsequence comprises a first attachment site (attP) and a seventhsequence, wherein the seventh sequence comprises a second attachmentsite (attB) wherein each of the first sequence, second sequence, thirdsequence, fourth sequence, fifth sequence, sixth sequence, and seventhsequence have a 5′ terminus and a 3′ terminus, and wherein the 3′terminus of the first sequence comprising the first inverted terminalrepeat gene sequence is adjacent to the 5′ terminus of the thirdsequence, the 3′ terminus of the third sequence is adjacent to the 5′terminus of the fourth sequence, the 3′ terminus of the fourth sequenceis adjacent to the 5′ terminus of the fifth sequence and the 3′ terminusof the fifth sequence is adjacent to the 5′ terminus of the secondsequence comprising a second inverted terminal repeat. In somealternatives, the gene encoding the double mutant of human dihydrofolatereductase comprises the DNA sequence:ATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACTTCCCCTGGCCACCGCTCAGGAATGAATCCAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAAGGAGCTCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAGAATGATTAA (SEQ ID NO: 2). In some alternatives, the doublemutant of human dihydrofolate reductase comprises the protein sequence:MVGSLNCIVA VSQNMGIGKN GDFPWPPLRN ESRYFQRMTT TSSVEGKQNL VIMGKKTWFSIPEKNRPLKG RINLVLSREL KEPPQGAHFL SRSLDDALKL TEQPELANKV DMVWIVGGSSVYKEAMNHPG HLKLFVTRIM QDFESDTFFP EIDLEKYKLL PEYPGVLSDV QEEKGIKYKFEVYEKND (SEQ ID NO: 3). In some alternatives, the gene deliverypolynucleotide is circular. In some alternatives, the gene deliverypolynucleotide is at least 1 kB to 5 kB. In some alternatives, the genedelivery polynucleotide is a minicircle. In some alternatives, thepromoter region comprises an EF1 promoter sequence. In somealternatives, the fourth sequence comprises one, two, three, four, orfive genes that encode proteins. In some alternatives, the fourthsequence is codon optimized to reduce the total GC/AT ratio of thefourth sequence. In some alternatives, the fourth sequence is optimizedby codon optimization for expression in humans. In some alternatives,the fourth sequence is a consensus sequence generated from a pluralityof nucleic acids that encode a plurality of related proteins. In somealternatives, the fourth sequence is a consensus sequence generated froma plurality of nucleic acids that encode a plurality of relatedproteins, such as a plurality of antibody binding domains, which arespecific for the same epitope. In some alternatives, the plurality ofrelated proteins comprise a plurality of antibody binding domains,wherein the plurality of antibody binding domains are specific for thesame epitope. In some alternatives, the fifth sequence is codonoptimized for expression in humans and/or to reduce the total GC/ATratio of the fifth sequence. In preferred alternatives, the fifthsequence is optimized by codon optimization for expression in humans. Insome alternatives, the protein is a protein for therapy. In somealternatives, the codon optimization and/or a consensus sequence isgenerated by comparing the variability of sequence and/or nucleobasesutilized in a plurality of related sequences. In some alternatives, theprotein comprises an antibody or a portion thereof, which may behumanized. In some alternatives, the double mutant of dihydrofolatereductase comprises amino acid mutations of L22F and F31S. In somealternatives, the T cells are precursor T cells. In some alternatives,the precursor T cells are hematopoietic stem cells.

In some alternatives, a method of generating engineered multiplexedT-cells for adoptive T-cell immunotherapy is provided, wherein themethod comprises providing a gene delivery polynucleotide, introducingthe gene delivery polynucleotide into a T-cell, providing a vectorencoding a Sleeping Beauty transposase, introducing the vector encodingthe Sleeping Beauty transposase into the T-cell, selecting the cellscomprising the gene delivery polynucleotide wherein selecting comprisesa first round of selection and a second round of selection, wherein thefirst round of selection comprises adding a selection reagent at a firstconcentration range and the second round of selection comprises addingthe selection reagent at a second concentration range, wherein thesecond concentration range is higher than the first concentration rangeand, wherein the second concentration range is at least 1.5 fold higherthan that of the first concentration range and isolating the T-cellsexpressing a phenotype under selective pressure. In some alternatives,the gene delivery polynucleotide comprises a first sequence, wherein thefirst sequence comprises a first inverted terminal repeat gene sequence,a second sequence, wherein the second sequence comprises a secondinverted terminal repeat gene sequence, a third sequence, wherein thethird sequence comprises a promoter region sequence, a fourth sequence,wherein the fourth sequence comprises at least one gene encoding aprotein, and wherein the fourth sequence is optimized, a fifth sequence,wherein the fifth sequence comprises at least one selectable markercassette encoding a double mutant of dihydrofolate reductase, whereinthe double mutant of dihydrofolate reductase has a 15,000 fold or about15,000 fold reduced affinity for methotrexate, wherein the methotrexatecan be used as a selection mechanism to selectively amplify cellstransduced with the gene delivery polynucleotide and wherein the fifthsequence is optimized, a sixth sequence, wherein the sixth sequencecomprises a first attachment site (attP) and a seventh sequence, whereinthe seventh sequence comprises a second attachment site (attB) whereineach of the first sequence, second sequence, third sequence, fourthsequence, fifth sequence, sixth sequence, and seventh sequence have a 5′terminus and a 3′ terminus, and wherein the 3′ terminus of the firstsequence comprising the first inverted terminal repeat gene sequence isadjacent to the 5′ terminus of the third sequence, the 3′ terminus ofthe third sequence is adjacent to the 5′ terminus of the fourthsequence, the 3′ terminus of the fourth sequence is adjacent to the 5′terminus of the fifth sequence and the 3′ terminus of the fifth sequenceis adjacent to the 5′ terminus of the second sequence comprising asecond inverted terminal repeat. In some alternatives, the gene encodingthe double mutant of human dihydrofolate reductase comprises the DNAsequence: ATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACTTCCCCTGGCCACCGCTCAGGAATGAATCCAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAAGGAGCTCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAGAATGATTAA (SEQ ID NO: 2). In some alternatives, the doublemutant of human dihydrofolate reductase comprises the protein sequence:MVGSLNCIVA VSQNMGIGKN GDFPWPPLRN ESRYFQRMTT TSSVEGKQNL VIMGKKTWFSIPEKNRPLKG RINLVLSREL KEPPQGAHFL SRSLDDALKL TEQPELANKV DMVWIVGGSSVYKEAMNHPG HLKLFVTRIM QDFESDTFFP EIDLEKYKLL PEYPGVLSDV QEEKGIKYKFEVYEKND (SEQ ID NO: 3). In some alternatives, the gene deliverypolynucleotide is circular. In some alternatives, the gene deliverypolynucleotide is at least 1 kB to 5 kB. In some alternatives, the genedelivery polynucleotide is a minicircle. In some alternatives, thepromoter region comprises an EF1 promoter sequence. In somealternatives, the fourth sequence comprises one, two, three, four, orfive genes that encode proteins. In some alternatives, the fourthsequence is codon optimized to reduce the total GC/AT ratio of thefourth sequence. In some alternatives, the fourth sequence is optimizedby codon optimization for expression in humans. In some alternatives,the fourth sequence is a consensus sequence generated from a pluralityof nucleic acids that encode a plurality of related proteins. In somealternatives, the fourth sequence is a consensus sequence generated froma plurality of nucleic acids that encode a plurality of relatedproteins, such as a plurality of antibody binding domains, which arespecific for the same epitope. In some alternatives, the plurality ofrelated proteins comprise a plurality of antibody binding domains,wherein the plurality of antibody binding domains are specific for thesame epitope. In some alternatives, the fifth sequence is codonoptimized to reduce the total GC/AT ratio of the fifth sequence. In somealternatives, the fifth sequence is optimized by codon optimization forexpression in humans. In some alternatives, the protein is a protein fortherapy. In some alternatives, the codon optimization and/or consensussequence is generated by comparing the variability of sequence and/ornucleobases utilized in a plurality of related sequences. In somealternatives, the protein comprises an antibody or a portion thereof,which may be humanized. In some alternatives, the double mutant ofdihydrofolate reductase comprises amino acid mutations of L22F and F31S.In some alternatives, the double mutant of dihydrofolate reductasecomprises amino acid mutations of L22F and F31S. In some alternatives,the introducing is performed by electroporation. In some alternatives,the selecting is performed by increasing selective pressure through theselective marker cassette. In some alternatives, the selection reagentcomprises an agent for selection. In some alternatives, the agent forselection is methotrexate. In some alternatives, the first concentrationrange is at least 50 nM-100 nM and the second concentration range is atleast 75 to 150 nM. In some alternatives, the first concentration is 50nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100 nM or any concentration that isbetween a range of concentrations defined by any two of theaforementioned concentrations, and the second concentration range is 75nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, or 150 nM orany concentration that is between a range of concentrations defined byany two of the aforementioned concentrations. In some alternatives, thefirst concentration range is at least 75 nM-150 nM and the secondconcentration range is at least 112.5 nM to 225 nM. In somealternatives, the first concentration is 75 nM, 85 nM, 95 nM, 105 nM,115 nM, 125 nM, 135 nM, 145 nM, or 150 nM or any concentration that isbetween a range of concentrations defined by any two of theaforementioned concentrations, and the second concentration range is 112nM, 122 nM, 132 nM, 142 nM, 152 nM, 162 nM, 172 nM, 182 nM, 192 nM, 202nM, 212 nM, or 225 nM or any concentration that is between a range ofconcentrations defined by any two of the aforementioned concentrations.In some alternatives, the first concentration range is at least 300nM-675 nM and the first concentration range is at least 450 nM to 1012nM. In some alternatives, the first concentration is 300 nM, 350 nM, 400nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, or 675 nM or anyconcentration that is between a range of concentrations defined by anytwo of the aforementioned concentrations, and the second concentrationrange is 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM,850 nM, 900 nM, 1000 nM, or 1012 nM or any concentration that is betweena range of concentrations defined by any two of the aforementionedconcentrations. In some alternatives, the first round of selectioncomprises exposing the T-cells to the selection agent for 2, 3, 4, 5, 6or 7 days before the second round of selection. In some alternatives,the second round of selection comprises exposing the T-cells to theselection agent for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or14 days or any time that is between a range of times defined by any twoof the aforementioned time points before isolation. In somealternatives, the T cells are precursor T cells. In some alternatives,the precursor T cells are hematopoietic stem cells.

In some alternatives, a method of increasing protein production in aT-cell is provided, wherein the method comprises providing apolynucleotide of, introducing the polynucleotide into a cell, providinga vector encoding a Sleeping Beauty transposase, introducing the vectorencoding the Sleeping Beauty transposase into the T-cell, selecting thecells comprising the gene delivery polynucleotide wherein selectingcomprises a first round of selection and a second round of selection,wherein the first round of selection comprises adding a selectionreagent at a first concentration range and the second round of selectioncomprises adding the selection reagent at a second concentration range,wherein the second concentration range is higher than the firstconcentration range and, wherein the second concentration range is atleast 1.5 fold higher than that of the first concentration range andisolating the cells expressing a phenotype under selective pressure. Insome alternatives, the gene delivery polynucleotide comprises a firstsequence, wherein the first sequence comprises a first inverted terminalrepeat gene sequence, a second sequence, wherein the second sequencecomprises a second inverted terminal repeat gene sequence, a thirdsequence, wherein the third sequence comprises a promoter regionsequence, a fourth sequence, wherein the fourth sequence comprises atleast one gene encoding a protein, and wherein the fourth sequence isoptimized, a fifth sequence, wherein the fifth sequence comprises atleast one selectable marker cassette encoding a double mutant ofdihydrofolate reductase, wherein the double mutant of dihydrofolatereductase has a 15,000 fold or about 15,000 fold reduced affinity formethotrexate, wherein the methotrexate can be used as a selectionmechanism to selectively amplify cells transduced with the gene deliverypolynucleotide and wherein the fifth sequence is optimized, a sixthsequence, wherein the sixth sequence comprises a first attachment site(attP) and a seventh sequence, wherein the seventh sequence comprises asecond attachment site (attB) wherein each of the first sequence, secondsequence, third sequence, fourth sequence, fifth sequence, sixthsequence, and seventh sequence have a 5′ terminus and a 3′ terminus, andwherein the 3′ terminus of the first sequence comprising the firstinverted terminal repeat gene sequence is adjacent to the 5′ terminus ofthe third sequence, the 3′ terminus of the third sequence is adjacent tothe 5′ terminus of the fourth sequence, the 3′ terminus of the fourthsequence is adjacent to the 5′ terminus of the fifth sequence and the 3′terminus of the fifth sequence is adjacent to the 5′ terminus of thesecond sequence comprising a second inverted terminal repeat. In somealternatives, the gene encoding the double mutant of human dihydrofolatereductase comprises the DNA sequence:ATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACTTCCCCTGGCCACCGCTCAGGAATGAATCCAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAAGGAGCTCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAGAATGATTAA (SEQ ID NO: 2). In some alternatives, the doublemutant of human dihydrofolate reductase comprises the protein sequence:MVGSLNCIVA VSQNMGIGKN GDFPWPPLRN ESRYFQRMTT TSSVEGKQNL VIMGKKTWFSIPEKNRPLKG RINLVLSREL KEPPQGAHFL SRSLDDALKL TEQPELANKV DMVWIVGGSSVYKEAMNHPG HLKLFVTRIM QDFESDTFFP EIDLEKYKLL PEYPGVLSDV QEEKGIKYKFEVYEKND (SEQ ID NO: 3). In some alternatives, the gene deliverypolynucleotide is circular. In some alternatives, the gene deliverypolynucleotide is at least 1 kB to 5 kB. In some alternatives, the genedelivery polynucleotide is a minicircle. In some alternatives, thepromoter region comprises an EF1 promoter sequence. In somealternatives, the fourth sequence comprises one, two, three, four, orfive genes that encode proteins. In some alternatives, the fourthsequence is codon optimized to reduce the total GC/AT ratio of thefourth sequence. In some alternatives, the fourth sequence is optimizedby codon optimization for expression in humans. In some alternatives,the fourth sequence is a consensus sequence generated from a pluralityof nucleic acids that encode a plurality of related proteins. In somealternatives, the fourth sequence is a consensus sequence generated froma plurality of nucleic acids that encode a plurality of relatedproteins, such as a plurality of antibody binding domains, which arespecific for the same epitope. In some alternatives, the plurality ofrelated proteins comprise a plurality of antibody binding domains,wherein the plurality of antibody binding domains are specific for thesame epitope. In some alternatives, the fifth sequence is codonoptimized to reduce the total GC/AT ratio of the fifth sequence. In somealternatives, the fifth sequence is optimized by codon optimization forexpression in humans. In some alternatives, the protein is a protein fortherapy. In some alternatives, the codon optimization and/or consensussequence is generated by comparing the variability of sequence and/ornucleobases utilized in a plurality of related sequences. In somealternatives, the protein comprises an antibody or a portion thereof,which may be humanized. In some alternatives, the double mutant ofdihydrofolate reductase comprises amino acid mutations of L22F and F31S.In some alternatives, the double mutant of dihydrofolate reductasecomprises amino acid mutations of L22F and F31S. In some alternatives,the introducing is performed by electroporation. In some alternatives,the selecting is performed by increasing selective pressure through theselective marker cassette. In some alternatives, the selection reagentcomprises an agent for selection. In some alternatives, the agent forselection is methotrexate. In some alternatives, the first concentrationrange is at least 50 nM-100 nM and the second concentration range is atleast 75 to 150 nM. In some alternatives, the first concentration is 50nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100 nM or any concentration that isbetween a range of concentrations defined by any two of theaforementioned concentrations, and the second concentration range is 75nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, or 150 nM orany concentration that is between a range of concentrations defined byany two of the aforementioned concentrations. In some alternatives, thefirst concentration range is at least 75 nM-150 nM and the secondconcentration range is at least 112.5 nM to 225 nM. In somealternatives, the first concentration is 75 nM, 85 nM, 95 nM, 105 nM,115 nM, 125 nM, 135 nM, 145 nM, 150 nM or any concentration that isbetween a range of concentrations defined by any two of theaforementioned concentrations, and the second concentration range is 112nM, 122 nM, 132 nM, 142 nM, 152 nM, 162 nM, 172 nM, 182 nM, 192 nM, 202nM, 212 nM, or 225 nM or any concentration that is between a range ofconcentrations defined by any two of the aforementioned concentrations.In some alternatives, the first concentration range is at least 300nM-675 nM and the first concentration range is at least 450 nM to 1012nM. In some alternatives, the first concentration is 300 nM, 350 nM, 400nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, or 675 nM or anyconcentration that is between a range of concentrations defined by anytwo of the aforementioned concentrations, and the second concentrationrange is 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM,850 nM, 900 nM, 1000 nM, or 1012 nM or any concentration that is betweena range of concentrations defined by any two of the aforementionedconcentrations. In some alternatives, the first round of selectioncomprises exposing the T-cells to the selection agent for 2, 3, 4, 5, 6or 7 days before the second round of selection. In some alternatives,the second round of selection comprises exposing the T-cells to theselection agent for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or14 days or any time that is between a range of times defined by any twoof the aforementioned time points before isolation. In somealternatives, the T cells are precursor T cells. In some alternatives,the precursor T cells are hematopoietic stem cells.

In some alternatives, an engineered multiplexed T-cell for adoptiveT-cell immunotherapy generated by any one of the methods of is provided.In some alternatives, the engineered multiplexed T-cells for adoptiveT-cell immunotherapy is generated by a method, wherein the methodcomprises providing a gene delivery polynucleotide, introducing the genedelivery polynucleotide into a T-cell, providing a vector encoding aSleeping Beauty transposase, introducing the vector encoding theSleeping Beauty transposase into the T-cell, selecting the cellscomprising the gene delivery polynucleotide wherein selecting comprisesa first round of selection and a second round of selection, wherein thefirst round of selection comprises adding a selection reagent at a firstconcentration range and the second round of selection comprises addingthe selection reagent at a second concentration range, wherein thesecond concentration range is higher than the first concentration rangeand, wherein the second concentration range is at least 1.5 fold higherthan that of the first concentration range and isolating the T-cellsexpressing a phenotype under selective pressure. In some alternatives,the gene delivery polynucleotide comprises a first sequence, wherein thefirst sequence comprises a first inverted terminal repeat gene sequence,a second sequence, wherein the second sequence comprises a secondinverted terminal repeat gene sequence, a third sequence, wherein thethird sequence comprises a promoter region sequence, a fourth sequence,wherein the fourth sequence comprises at least one gene encoding aprotein, and wherein the fourth sequence is optimized, a fifth sequence,wherein the fifth sequence comprises at least one selectable markercassette encoding a double mutant of dihydrofolate reductase, whereinthe double mutant of dihydrofolate reductase has a 15,000 fold or about15,000 fold reduced affinity for methotrexate, wherein the methotrexatecan be used as a selection mechanism to selectively amplify cellstransduced with the gene delivery polynucleotide and wherein the fifthsequence is optimized, a sixth sequence, wherein the sixth sequencecomprises a first attachment site (attP) and a seventh sequence, whereinthe seventh sequence comprises a second attachment site (attB) whereineach of the first sequence, second sequence, third sequence, fourthsequence, fifth sequence, sixth sequence, and seventh sequence have a 5′terminus and a 3′ terminus, and wherein the 3′ terminus of the firstsequence comprising the first inverted terminal repeat gene sequence isadjacent to the 5′ terminus of the third sequence, the 3′ terminus ofthe third sequence is adjacent to the 5′ terminus of the fourthsequence, the 3′ terminus of the fourth sequence is adjacent to the 5′terminus of the fifth sequence and the 3′ terminus of the fifth sequenceis adjacent to the 5′ terminus of the second sequence comprising asecond inverted terminal repeat. In some alternatives, the gene encodingthe double mutant of human dihydrofolate reductase comprises the DNAsequence: ATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACTTCCCCTGGCCACCGCTCAGGAATGAATCCAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAAGGAGCTCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAGAATGATTAA (SEQ ID NO: 2). In some alternatives, the doublemutant of human dihydrofolate reductase comprises the protein sequence:MVGSLNCIVA VSQNMGIGKN GDFPWPPLRN ESRYFQRMTT TSSVEGKQNL VIMGKKTWFSIPEKNRPLKG RINLVLSREL KEPPQGAHFL SRSLDDALKL TEQPELANKV DMVWIVGGSSVYKEAMNHPG HLKLFVTRIM QDFESDTFFP EIDLEKYKLL PEYPGVLSDV QEEKGIKYKFEVYEKND (SEQ ID NO: 3). In some alternatives, the gene deliverypolynucleotide is circular. In some alternatives, the gene deliverypolynucleotide is at least 1 kB to 5 kB. In some alternatives, the genedelivery polynucleotide is a minicircle. In some alternatives, thepromoter region comprises an EF1 promoter sequence. In somealternatives, the fourth sequence comprises one, two, three, four, orfive genes that encode proteins. In some alternatives, the fourthsequence is codon optimized to reduce the total GC/AT ratio of thefourth sequence. In some alternatives, the fourth sequence is optimizedby codon optimization for expression in humans. In some alternatives,the fourth sequence is a consensus sequence generated from a pluralityof nucleic acids that encode a plurality of related proteins. In somealternatives, the fourth sequence is a consensus sequence generated froma plurality of nucleic acids that encode a plurality of relatedproteins, such as a plurality of antibody binding domains, which arespecific for the same epitope. In some alternatives, the plurality ofrelated proteins comprise a plurality of antibody binding domains,wherein the plurality of antibody binding domains are specific for thesame epitope. In some alternatives, the fifth sequence is codonoptimized to reduce the total GC/AT ratio of the fifth sequence. In somealternatives, the fifth sequence is optimized by codon optimization forexpression in humans. In some alternatives, the protein is a protein fortherapy. In some alternatives, the codon optimization and/or consensussequence is generated by comparing the variability of sequence and/ornucleobases utilized in a plurality of related sequences. In somealternatives, the protein comprises an antibody or a portion thereof,which may be humanized. In some alternatives, the double mutant ofdihydrofolate reductase comprises amino acid mutations of L22F and F31S.In some alternatives, the double mutant of dihydrofolate reductasecomprises amino acid mutations of L22F and F31S. In some alternatives,the introducing is performed by electroporation. In some alternatives,the selecting is performed by increasing selective pressure through theselective marker cassette. In some alternatives, the selection reagentcomprises an agent for selection. In some alternatives, the agent forselection is methotrexate. In some alternatives, the first concentrationrange is at least 50 nM-100 nM and the second concentration range is atleast 75 to 150 nM. In some alternatives, the first concentration is 50nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100 nM or any concentration that isbetween a range of concentrations defined by any two of theaforementioned concentrations, and the second concentration range is 75nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, or 150 nM orany concentration that is between a range of concentrations defined byany two of the aforementioned concentrations. In some alternatives, thefirst concentration range is at least 75 nM-150 nM and the secondconcentration range is at least 112.5 nM to 225 nM. In somealternatives, the first concentration is 75 nM, 85 nM, 95 nM, 105 nM,115 nM, 125 nM, 135 nM, 145 nM, or 150 nM or any concentration that isbetween a range of concentrations defined by any two of theaforementioned concentrations, and the second concentration range is 112nM, 122 nM, 132 nM, 142 nM, 152 nM, 162 nM, 172 nM, 182 nM, 192 nM, 202nM, 212 nM, or 225 nM or any concentration that is between a range ofconcentrations defined by any two of the aforementioned concentrations.In some alternatives, the first concentration range is at least 300nM-675 nM and the first concentration range is at least 450 nM to 1012nM. In some alternatives, the first concentration is 300 nM, 350 nM, 400nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, or 675 nM or anyconcentration that is between a range of concentrations defined by anytwo of the aforementioned concentrations, and the second concentrationrange is 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM,850 nM, 900 nM, 1000 nM, or 1012 nM or any concentration that is betweena range of concentrations defined by any two of the aforementionedconcentrations. In some alternatives, the first round of selectioncomprises exposing the T-cells to the selection agent for 2, 3, 4, 5, 6or 7 days before the second round of selection. In some alternatives,the second round of selection comprises exposing the T-cells to theselection agent for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or14 days or any time that is between a range of times defined by any twoof the aforementioned time points before isolation. In somealternatives, the gene delivery polynucleotide comprises a firstsequence, wherein the first sequence comprises a first inverted terminalrepeat gene sequence, a second sequence, wherein the second sequencecomprises a second inverted terminal repeat gene sequence, a thirdsequence, wherein the third sequence comprises a promoter regionsequence, a fourth sequence, wherein the fourth sequence comprises atleast one gene encoding a protein, and wherein the fourth sequence isoptimized, a fifth sequence, wherein the fifth sequence comprises atleast one selectable marker cassette encoding a double mutant ofdihydrofolate reductase, wherein the double mutant of dihydrofolatereductase has a 15,000 fold or about 15,000 fold reduced affinity formethotrexate, wherein the methotrexate can be used as a selectionmechanism to selectively amplify cells transduced with the gene deliverypolynucleotide and wherein the fifth sequence is optimized, a sixthsequence, wherein the sixth sequence comprises a first attachment site(attP) and a seventh sequence, wherein the seventh sequence comprises asecond attachment site (attB) wherein each of the first sequence, secondsequence, third sequence, fourth sequence, fifth sequence, sixthsequence, and seventh sequence have a 5′ terminus and a 3′ terminus, andwherein the 3′ terminus of the first sequence comprising the firstinverted terminal repeat gene sequence is adjacent to the 5′ terminus ofthe third sequence, the 3′ terminus of the third sequence is adjacent tothe 5′ terminus of the fourth sequence, the 3′ terminus of the fourthsequence is adjacent to the 5′ terminus of the fifth sequence and the 3′terminus of the fifth sequence is adjacent to the 5′ terminus of thesecond sequence comprising a second inverted terminal repeat. In somealternatives, the gene encoding the double mutant of human dihydrofolatereductase comprises the DNA sequence:ATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACTTCCCCTGGCCACCGCTCAGGAATGAATCCAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAAGGAGCTCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAGAATGATTAA (SEQ ID NO: 2). In some alternatives, the doublemutant of human dihydrofolate reductase comprises the protein sequence:MVGSLNCIVA VSQNMGIGKN GDFPWPPLRN ESRYFQRMTT TSSVEGKQNL VIMGKKTWFSIPEKNRPLKG RINLVLSREL KEPPQGAHFL SRSLDDALKL TEQPELANKV DMVWIVGGSSVYKEAMNHPG HLKLFVTRIM QDFESDTFFP EIDLEKYKLL PEYPGVLSDV QEEKGIKYKFEVYEKND (SEQ ID NO: 3). In some alternatives, the gene deliverypolynucleotide is circular. In some alternatives, the gene deliverypolynucleotide is at least 1 kB to 5 kB. In some alternatives, the genedelivery polynucleotide is a minicircle. In some alternatives, thepromoter region comprises an EF1 promoter sequence. In somealternatives, the fourth sequence comprises one, two, three, four, orfive genes that encode proteins. In some alternatives, the fourthsequence is codon optimized to reduce the total GC/AT ratio of thefourth sequence. In some alternatives, the fourth sequence is optimizedby codon optimization for expression in humans. In some alternatives,the fourth sequence is a consensus sequence generated from a pluralityof nucleic acids that encode a plurality of related proteins. In somealternatives, the fourth sequence is a consensus sequence generated froma plurality of nucleic acids that encode a plurality of relatedproteins, such as a plurality of antibody binding domains, which arespecific for the same epitope. In some alternatives, the plurality ofrelated proteins comprise a plurality of antibody binding domains,wherein the plurality of antibody binding domains are specific for thesame epitope. In some alternatives, the fifth sequence is codonoptimized to reduce the total GC/AT ratio of the fifth sequence. In somealternatives, the fifth sequence is optimized by codon optimization forexpression in humans. In some alternatives, the protein is a protein fortherapy. In some alternatives, the codon optimization and/or consensussequence is generated by comparing the variability of sequence and/ornucleobases utilized in a plurality of related sequences. In somealternatives, the protein comprises an antibody or a portion thereof,which may be humanized. In some alternatives, the double mutant ofdihydrofolate reductase comprises amino acid mutations of L22F and F31S.In some alternatives, the introducing is performed by electroporation.In some alternatives, the selecting is performed by increasing selectivepressure through the selective marker cassette. In some alternatives,the selection reagent comprises an agent for selection. In somealternatives, the agent for selection is methotrexate. In somealternatives, the first concentration range is at least 50 nM-100 nM andthe second concentration range is at least 75 to 150 nM. In somealternatives, the first concentration is 50 nM, 60 nM, 70 nM, 80 nM, 90nM, or 100 nM or any concentration that is between a range ofconcentrations defined by any two of the aforementioned concentrations,and the second concentration range is 75 nM, 80 nM, 90 nM, 100 nM, 110nM, 120 nM, 130 nM, 140 nM, or 150 nM or any concentration that isbetween a range of concentrations defined by any two of theaforementioned concentrations. In some alternatives, the firstconcentration range is at least 75 nM-150 nM and the secondconcentration range is at least 112.5 nM to 225 nM. In somealternatives, the first concentration is 75 nM, 85 nM, 95 nM, 105 nM,115 nM, 125 nM, 135 nM, 145 nM, or 150 nM or any concentration that isbetween a range of concentrations defined by any two of theaforementioned concentrations, and the second concentration range is 112nM, 122 nM, 132 nM, 142 nM, 152 nM, 162 nM, 172 nM, 182 nM, 192 nM, 202nM, 212 nM, or 225 nM or any concentration that is between a range ofconcentrations defined by any two of the aforementioned concentrations.In some alternatives, the first concentration range is at least 300nM-675 nM and the first concentration range is at least 450 nM to 1012nM. In some alternatives, the first concentration is 300 nM, 350 nM, 400nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, or 675 nM or anyconcentration that is between a range of concentrations defined by anytwo of the aforementioned concentrations, and the second concentrationrange is 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM,850 nM, 900 nM, 1000 nM, or 1012 nM or any concentration that is betweena range of concentrations defined by any two of the aforementionedconcentrations. In some alternatives, the first round of selectioncomprises exposing the T-cells to the selection agent for 2, 3, 4, 5, 6or 7 days before the second round of selection. In some alternatives,the second round of selection comprises exposing the T-cells to theselection agent for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or14 days or any time that is between a range of times defined by any twoof the aforementioned time points before isolation. In somealternatives, the T cells are precursor T cells. In some alternatives,the precursor T cells are hematopoietic stem cells.

In some alternatives, a method of treating, inhibiting, or amelioratingcancer or a disease in a subject is provided, wherein the methodcomprises administering to the subject the modified or engineeredmultiplexed T-cell generated as described below. In some alternatives,the engineered multiplexed T-cells for adoptive T-cell immunotherapy isgenerated by a method, wherein the method comprises providing a genedelivery polynucleotide, introducing the gene delivery polynucleotideinto a T-cell, providing a vector encoding a Sleeping Beautytransposase, introducing the vector encoding the Sleeping Beautytransposase into the T-cell, selecting the cells comprising the genedelivery polynucleotide wherein selecting comprises a first round ofselection and a second round of selection, wherein the first round ofselection comprises adding a selection reagent at a first concentrationrange and the second round of selection comprises adding the selectionreagent at a second concentration range, wherein the secondconcentration range is higher than the first concentration range and,wherein the second concentration range is at least 1.5 fold higher thanthat of the first concentration range and isolating the T-cellsexpressing a phenotype under selective pressure. In some alternatives,the gene delivery polynucleotide comprises a first sequence, wherein thefirst sequence comprises a first inverted terminal repeat gene sequence,a second sequence, wherein the second sequence comprises a secondinverted terminal repeat gene sequence, a third sequence, wherein thethird sequence comprises a promoter region sequence, a fourth sequence,wherein the fourth sequence comprises at least one gene encoding aprotein, and wherein the fourth sequence is optimized, a fifth sequence,wherein the fifth sequence comprises at least one selectable markercassette encoding a double mutant of dihydrofolate reductase, whereinthe double mutant of dihydrofolate reductase has a 15,000 fold or about15,000 fold reduced affinity for methotrexate, wherein the methotrexatecan be used as a selection mechanism to selectively amplify cellstransduced with the gene delivery polynucleotide and wherein the fifthsequence is optimized, a sixth sequence, wherein the sixth sequencecomprises a first attachment site (attP) and a seventh sequence, whereinthe seventh sequence comprises a second attachment site (attB) whereineach of the first sequence, second sequence, third sequence, fourthsequence, fifth sequence, sixth sequence, and seventh sequence have a 5′terminus and a 3′ terminus, and wherein the 3′ terminus of the firstsequence comprising the first inverted terminal repeat gene sequence isadjacent to the 5′ terminus of the third sequence, the 3′ terminus ofthe third sequence is adjacent to the 5′ terminus of the fourthsequence, the 3′ terminus of the fourth sequence is adjacent to the 5′terminus of the fifth sequence and the 3′ terminus of the fifth sequenceis adjacent to the 5′ terminus of the second sequence comprising asecond inverted terminal repeat. In some alternatives, the gene encodingthe double mutant of human dihydrofolate reductase comprises the DNAsequence: ATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACTTCCCCTGGCCACCGCTCAGGAATGAATCCAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAAGGAGCTCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAGAATGATTAA (SEQ ID NO: 2). In some alternatives, the doublemutant of human dihydrofolate reductase comprises the protein sequence:MVGSLNCIVA VSQNMGIGKN GDFPWPPLRN ESRYFQRMTT TSSVEGKQNL VIMGKKTWFSIPEKNRPLKG RINLVLSREL KEPPQGAHFL SRSLDDALKL TEQPELANKV DMVWIVGGSSVYKEAMNHPG HLKLFVTRIM QDFESDTFFP EIDLEKYKLL PEYPGVLSDV QEEKGIKYKFEVYEKND (SEQ ID NO: 3). In some alternatives, the gene deliverypolynucleotide is circular. In some alternatives, the gene deliverypolynucleotide is a minicircle. In some alternatives, the gene deliverypolynucleotide is at least 1 kB to 5 kB. In some alternatives, thepromoter region comprises an EF1 promoter sequence. In somealternatives, the fourth sequence comprises one, two, three, four, orfive genes that encode proteins. In some alternatives, the fourthsequence is codon optimized to reduce the total GC/AT ratio of thefourth sequence. In some alternatives, the fourth sequence is optimizedby codon optimization for expression in humans. In some alternatives,the fourth sequence is a consensus sequence generated from a pluralityof nucleic acids that encode a plurality of related proteins. In somealternatives, the fourth sequence is a consensus sequence generated froma plurality of nucleic acids that encode a plurality of relatedproteins, such as a plurality of antibody binding domains, which arespecific for the same epitope. In some alternatives, the plurality ofrelated proteins comprise a plurality of antibody binding domains,wherein the plurality of antibody binding domains are specific for thesame epitope. In some alternatives, the fifth sequence is codonoptimized to reduce the total GC/AT ratio of the fifth sequence. In somealternatives, the fifth sequence is optimized by codon optimization forexpression in humans. In some alternatives, the protein is a protein fortherapy. In some alternatives, the codon optimization and/or consensussequence is generated by comparing the variability of sequence and/ornucleobases utilized in a plurality of related sequences. In somealternatives, the protein comprises an antibody or a portion thereof,which may be humanized. In some alternatives, the double mutant ofdihydrofolate reductase comprises amino acid mutations of L22F and F31S.In some alternatives, the double mutant of dihydrofolate reductasecomprises amino acid mutations of L22F and F31S. In some alternatives,the T cells are precursor T cells. In some alternatives, the precursor Tcells are hematopoietic stem cells. In some alternatives, theintroducing is performed by electroporation. In some alternatives, theselecting is performed by increasing selective pressure through theselective marker cassette. In some alternatives, the selection reagentcomprises an agent for selection. In some alternatives, the agent forselection is methotrexate. In some alternatives, the first concentrationrange is at least 50 nM-100 nM and the second concentration range is atleast 75 to 150 nM. In some alternatives, the first concentration is 50nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100 nM or any concentration that isbetween a range of concentrations defined by any two of theaforementioned concentrations, and the second concentration range is 75nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, or 150 nM orany concentration that is between a range of concentrations defined byany two of the aforementioned concentrations. In some alternatives, thefirst concentration range is at least 75 nM-150 nM and the secondconcentration range is at least 112.5 nM to 225 nM. In somealternatives, the first concentration is 75 nM, 85 nM, 95 nM, 105 nM,115 nM, 125 nM, 135 nM, 145 nM, or 150 nM or any concentration that isbetween a range of concentrations defined by any two of theaforementioned concentrations, and the second concentration range is 112nM, 122 nM, 132 nM, 142 nM, 152 nM, 162 nM, 172 nM, 182 nM, 192 nM, 202nM, 212 nM, or 225 nM or any concentration that is between a range ofconcentrations defined by any two of the aforementioned concentrations.In some alternatives, the first concentration range is at least 300nM-675 nM and the first concentration range is at least 450 nM to 1012nM. In some alternatives, the first concentration is 300 nM, 350 nM, 400nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, or 675 nM or anyconcentration that is between a range of concentrations defined by anytwo of the aforementioned concentrations, and the second concentrationrange is 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM,850 nM, 900 nM, 1000 nM, or 1012 nM or any concentration that is betweena range of concentrations defined by any two of the aforementionedconcentrations. In some alternatives, the first round of selectioncomprises exposing the T-cells to the selection agent for 2, 3, 4, 5, 6or 7 days before the second round of selection. In some alternatives,the second round of selection comprises exposing the T-cells to theselection agent for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or14 days or any time that is between a range of times defined by any twoof the aforementioned time points before isolation. In somealternatives, the subject is human.

In some alternatives, a method of generating engineered multiplexedT-cells for adoptive T-cell immunotherapy is provided, wherein themethod comprises providing a gene delivery polynucleotide, introducingthe gene delivery polynucleotide into a T-cell, providing a vectorencoding a Sleeping Beauty transposase, introducing the vector encodingthe Sleeping Beauty transposase into the T-cell, selecting the cellscomprising the gene delivery polynucleotide wherein selecting comprisesa first round of selection and a second round of selection, wherein thefirst round of selection comprises adding a selection reagent at a firstconcentration range and the second round of selection comprises addingthe selection reagent at a second concentration range, wherein thesecond concentration range is higher than the first concentration rangeand, wherein the second concentration range is at least 1.5 fold higherthan that of the first concentration range and isolating the T-cellsexpressing a phenotype under selective pressure. In some alternatives,the gene delivery polynucleotide comprises a first sequence, wherein thefirst sequence comprises a first inverted terminal repeat gene sequence,a second sequence, wherein the second sequence comprises a secondinverted terminal repeat gene sequence, a third sequence, wherein thethird sequence comprises a promoter region sequence, a fourth sequence,wherein the fourth sequence comprises at least one gene encoding aprotein, and wherein the fourth sequence is optimized, a fifth sequence,wherein the fifth sequence comprises at least one selectable markercassette encoding a double mutant of dihydrofolate reductase, whereinthe double mutant of dihydrofolate reductase has a 15,000 fold or about15,000 fold reduced affinity for methotrexate, wherein the methotrexatecan be used as a selection mechanism to selectively amplify cellstransduced with the gene delivery polynucleotide and wherein the fifthsequence is optimized, a sixth sequence, wherein the sixth sequencecomprises a first attachment site (attP) and a seventh sequence, whereinthe seventh sequence comprises a second attachment site (attB) whereineach of the first sequence, second sequence, third sequence, fourthsequence, fifth sequence, sixth sequence, and seventh sequence have a 5′terminus and a 3′ terminus, and wherein the 3′ terminus of the firstsequence comprising the first inverted terminal repeat gene sequence isadjacent to the 5′ terminus of the third sequence, the 3′ terminus ofthe third sequence is adjacent to the 5′ terminus of the fourthsequence, the 3′ terminus of the fourth sequence is adjacent to the 5′terminus of the fifth sequence and the 3′ terminus of the fifth sequenceis adjacent to the 5′ terminus of the second sequence comprising asecond inverted terminal repeat. In some alternatives, the gene encodingthe double mutant of human dihydrofolate reductase comprises the DNAsequence: ATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACTTCCCCTGGCCACCGCTCAGGAATGAATCCAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAAGGAGCTCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAGAATGATTAA (SEQ ID NO: 2). In some alternatives, the doublemutant of human dihydrofolate reductase comprises the protein sequence:MVGSLNCIVA VSQNMGIGKN GDFPWPPLRN ESRYFQRMTT TSSVEGKQNL VIMGKKTWFSIPEKNRPLKG RINLVLSREL KEPPQGAHFL SRSLDDALKL TEQPELANKV DMVWIVGGSSVYKEAMNHPG HLKLFVTRIM QDFESDTFFP EIDLEKYKLL PEYPGVLSDV QEEKGIKYKFEVYEKND (SEQ ID NO: 3). In some alternatives, the gene deliverypolynucleotide is circular. In some alternatives, the gene deliverypolynucleotide is at least 1 kB to 5 kB. In some alternatives, the genedelivery polynucleotide is a minicircle. In some alternatives, thepromoter region comprises an EF1 promoter sequence. In somealternatives, the fourth sequence comprises one, two, three, four, orfive genes that encode proteins. In some alternatives, the fourthsequence is codon optimized to reduce the total GC/AT ratio of thefourth sequence. In some alternatives, the fourth sequence is optimizedby codon optimization for expression in humans. In some alternatives,the fourth sequence is a consensus sequence generated from a pluralityof nucleic acids that encode a plurality of related proteins. In somealternatives, the fourth sequence is a consensus sequence generated froma plurality of nucleic acids that encode a plurality of relatedproteins, such as a plurality of antibody binding domains, which arespecific for the same epitope. In some alternatives, the plurality ofrelated proteins comprise a plurality of antibody binding domains,wherein the plurality of antibody binding domains are specific for thesame epitope. In some alternatives, the fifth sequence is codonoptimized to reduce the total GC/AT ratio of the fifth sequence. In somealternatives, the fifth sequence is optimized by codon optimization forexpression in humans. In some alternatives, the protein is a protein fortherapy. In some alternatives, the codon optimization and/or consensussequence is generated by comparing the variability of sequence and/ornucleobases utilized in a plurality of related sequences. In somealternatives, the protein comprises an antibody or a portion thereof,which may be humanized. In some alternatives, the double mutant ofdihydrofolate reductase comprises amino acid mutations of L22F and F31S.In some alternatives, the double mutant of dihydrofolate reductasecomprises amino acid mutations of L22F and F31S. In some alternatives,the introducing is performed by electroporation. In some alternatives,the selecting is performed by increasing selective pressure through theselective marker cassette. In some alternatives, the selection reagentcomprises an agent for selection. In some alternatives, the agent forselection is methotrexate. In some alternatives, the first concentrationrange is at least 50 nM-100 nM and the second concentration range is atleast 75 to 150 nM. In some alternatives, the first concentration is 50nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100 nM or any concentration that isbetween a range of concentrations defined by any two of theaforementioned concentrations, and the second concentration range is 75nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, or 150 nM orany concentration that is between a range of concentrations defined byany two of the aforementioned concentrations. In some alternatives, thefirst concentration range is at least 75 nM-150 nM and the secondconcentration range is at least 112.5 nM to 225 nM. In somealternatives, the first concentration is 75 nM, 85 nM, 95 nM, 105 nM,115 nM, 125 nM, 135 nM, 145 nM, or 150 nM or any concentration that isbetween a range of concentrations defined by any two of theaforementioned concentrations, and the second concentration range is 112nM, 122 nM, 132 nM, 142 nM, 152 nM, 162 nM, 172 nM, 182 nM, 192 nM, 202nM, 212 nM, or 225 nM or any concentration that is between a range ofconcentrations defined by any two of the aforementioned concentrations.In some alternatives, the first concentration range is at least 300nM-675 nM and the first concentration range is at least 450 nM to 1012nM. In some alternatives, the first concentration is 300 nM, 350 nM, 400nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, or 675 nM or anyconcentration that is between a range of concentrations defined by anytwo of the aforementioned concentrations, and the second concentrationrange is 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM,850 nM, 900 nM, 1000 nM, or 1012 nM or any concentration that is betweena range of concentrations defined by any two of the aforementionedconcentrations. In some alternatives, the first round of selectioncomprises exposing the T-cells to the selection agent for 2, 3, 4, 5, 6or 7 days before the second round of selection. In some alternatives,the second round of selection comprises exposing the T-cells to theselection agent for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or14 days or any time that is between a range of times defined by any twoof the aforementioned time points before isolation. In somealternatives, the T cells comprise precursor T cells. In somealternatives, the precursor T cells are hematopoietic stem cells.

In some alternatives, a method of generating engineered cells foradoptive T-cell immunotherapy comprising, providing a gene deliverypolynucleotide, introducing the gene delivery polynucleotide into aprecursor T cell, providing a vector encoding a Sleeping Beautytransposase, introducing the vector encoding the Sleeping Beautytransposase into the precursor T cell, selecting the precursor T cellscomprising the gene delivery polynucleotide; wherein selecting comprisesa first round of selection and a second round of selection, wherein thefirst round of selection comprises adding a selection reagent at a firstconcentration range and the second round of selection comprises addingthe selection reagent at a second concentration range, wherein thesecond concentration range is higher than the first concentration rangeand, wherein the second concentration range is at least 1.5 fold higherthan that of the first concentration range and isolating the precursorT-cells expressing a phenotype under selective pressure. In somealternatives, the gene delivery polynucleotide is for stable insertionof a nucleic acid into an oligonucleotide wherein the nucleic acid forinsertion is flanked by inverted terminal repeat gene sequences in thegene delivery polynucleotide and wherein the gene deliverypolynucleotide is selectable, wherein the gene delivery polynucleotidecomprises a first sequence, wherein the first sequence comprises a firstinverted terminal repeat gene sequence, a second sequence, wherein thesecond sequence comprises a second inverted terminal repeat genesequence, a third sequence, wherein the third sequence comprises apromoter region sequence, a fourth sequence, wherein the fourth sequencecomprises at least one gene, wherein the at least one gene encodes aprotein or encodes a sequence for mRNA transcription, and wherein thefourth sequence is optimized, a fifth sequence, wherein the fifthsequence comprises at least one selectable marker cassette encoding adouble mutant of dihydrofolate reductase, wherein the double mutant ofdihydrofolate reductase has a 15,000 fold or about 15,000 fold reducedaffinity for methotrexate, wherein the methotrexate can be used toselect for cells transduced with the gene delivery polynucleotide, toenhance the ratio of cells expressing the at least one gene and whereinthe fifth sequence is optimized, a sixth sequence, wherein the sixthsequence comprises a first attachment site (attP) and a seventhsequence, wherein the seventh sequence comprises a second attachmentsite (attB); wherein each of the first sequence, second sequence, thirdsequence, fourth sequence, fifth sequence, sixth sequence, and seventhsequence have a 5′ terminus and a 3′ terminus, and wherein the 3′terminus of the first sequence comprising the first inverted terminalrepeat gene sequence is adjacent to the 5′ terminus of the thirdsequence, the 3′ terminus of the third sequence is adjacent to the 5′terminus of the fourth sequence, the 3′ terminus of the fourth sequenceis adjacent to the 5′ terminus of the fifth sequence and the 3′ terminusof the fifth sequence is adjacent to the 5′ terminus of the secondsequence comprising a second inverted terminal repeat. In somealternatives, the gene delivery polynucleotide is circular. In somealternatives, the gene delivery polynucleotide is at least 1 kB to 5 kB.In some alternatives, the promoter region comprises an EF1 promotersequence. In some alternatives, the fourth sequence comprises one, two,three, four, or five genes that encode proteins. In some alternatives,the fourth sequence is codon optimized to reduce the total GC/AT ratioof the fourth sequence. In some alternatives, the fourth sequence isoptimized by codon optimization for expression in humans. In somealternatives, the fourth sequence is a consensus sequence generated froma plurality of nucleic acids that encode a plurality of relatedproteins. In some alternatives, the fourth sequence is a consensussequence generated from a plurality of nucleic acids that encode aplurality of related proteins, such as a plurality of antibody bindingdomains, which are specific for the same epitope. In some alternatives,the plurality of related proteins comprise a plurality of antibodybinding domains, wherein the plurality of antibody binding domains arespecific for the same epitope. In some alternatives, the fifth sequenceis codon optimized to reduce the total GC/AT ratio of the fifthsequence. In some alternatives, the fifth sequence is optimized by codonoptimization for expression in humans. In some alternatives, the codonoptimization and/or consensus sequence is generated by comparing thevariability of sequence and/or nucleobases utilized in a plurality ofrelated sequences. In some alternatives, the protein is a protein fortherapy. In some alternatives, the protein comprises an antibody or aportion thereof, which may be humanized. In some alternatives, thedouble mutant of dihydrofolate reductase comprises amino acid mutationsof L22F and F31S. In some alternatives, the gene delivery polynucleotideis a minicircle. In some alternatives, the introducing is performed byelectroporation. In some alternatives, the selecting is performed byincreasing selective pressure through the selective marker cassette. Insome alternatives, the selection reagent comprises an agent forselection. In some alternatives, the agent for selection ismethotrexate. In some alternatives, the first concentration range is atleast 50 nM-100 nM and the second concentration range is at least 75 to150 nM. In some alternatives, the first concentration range is at least75 nM-150 nM and the second concentration range is at least 112.5 nM to225 nM. In some alternatives, the first concentration range is at least300 nM-675 nM and the first concentration range is at least 450 nM to1012 nM. In some alternatives, the first round of selection comprisesexposing the T-cells to the selection agent for 2, 3, 4, 5, 6 or 7 daysbefore the second round of selection. In some alternatives, the secondround of selection comprises exposing the T-cells to the selection agentfor at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or anytime that is between a range of times defined by any two of theaforementioned time points before isolation. In some alternatives, the Tcell precursor is a hematopoietic stem cell.

In some alternatives, a method of increasing protein production in aprecursor T-cell is provided wherein the method comprises providing apolynucleotide, introducing the polynucleotide into a cell, providing avector encoding a Sleeping Beauty transposase; introducing the vectorencoding the Sleeping Beauty transposase into the precursor T-cell,selecting the precursor T cells comprising the gene deliverypolynucleotide, wherein selecting comprises a first round of selectionand a second round of selection, wherein the first round of selectioncomprises adding a selection reagent at a first concentration range andthe second round of selection comprises adding the selection reagent ata second concentration range, wherein the second concentration range ishigher than the first concentration range and, wherein the secondconcentration range is at least 1.5 fold higher than that of the firstconcentration range and isolating the precursor T cells expressing aphenotype under selective pressure. In some alternatives, the genedelivery polynucleotide is for stable insertion of a nucleic acid intoan oligonucleotide wherein the nucleic acid for insertion is flanked byinverted terminal repeat gene sequences in the gene deliverypolynucleotide and wherein the gene delivery polynucleotide isselectable, wherein the gene delivery polynucleotide comprises a firstsequence, wherein the first sequence comprises a first inverted terminalrepeat gene sequence, a second sequence, wherein the second sequencecomprises a second inverted terminal repeat gene sequence, a thirdsequence, wherein the third sequence comprises a promoter regionsequence, a fourth sequence, wherein the fourth sequence comprises atleast one gene, wherein the at least one gene encodes a protein orencodes a sequence for mRNA transcription, and wherein the fourthsequence is optimized, a fifth sequence, wherein the fifth sequencecomprises at least one selectable marker cassette encoding a doublemutant of dihydrofolate reductase, wherein the double mutant ofdihydrofolate reductase has a 15,000 fold or about 15,000 fold reducedaffinity for methotrexate, wherein the methotrexate can be used toselect for cells transduced with the gene delivery polynucleotide, toenhance the ratio of cells expressing the at least one gene and whereinthe fifth sequence is optimized, a sixth sequence, wherein the sixthsequence comprises a first attachment site (attP) and a seventhsequence, wherein the seventh sequence comprises a second attachmentsite (attB); wherein each of the first sequence, second sequence, thirdsequence, fourth sequence, fifth sequence, sixth sequence, and seventhsequence have a 5′ terminus and a 3′ terminus, and wherein the 3′terminus of the first sequence comprising the first inverted terminalrepeat gene sequence is adjacent to the 5′ terminus of the thirdsequence, the 3′ terminus of the third sequence is adjacent to the 5′terminus of the fourth sequence, the 3′ terminus of the fourth sequenceis adjacent to the 5′ terminus of the fifth sequence and the 3′ terminusof the fifth sequence is adjacent to the 5′ terminus of the secondsequence comprising a second inverted terminal repeat. In somealternatives, the gene delivery polynucleotide is circular. In somealternatives, the gene delivery polynucleotide is at least 1 kB to 5 kB.In some alternatives, the promoter region comprises an EF1 promotersequence. In some alternatives, the fourth sequence comprises one, two,three, four, or five genes that encode proteins. In some alternatives,the fourth sequence is codon optimized to reduce the total GC/AT ratioof the fourth sequence. In some alternatives, the fourth sequence isoptimized by codon optimization for expression in humans. In somealternatives, the fourth sequence is a consensus sequence generated froma plurality of nucleic acids that encode a plurality of relatedproteins. In some alternatives, the fourth sequence is a consensussequence generated from a plurality of nucleic acids that encode aplurality of related proteins, such as a plurality of antibody bindingdomains, which are specific for the same epitope. In some alternatives,the plurality of related proteins comprise a plurality of antibodybinding domains, wherein the plurality of antibody binding domains arespecific for the same epitope. In some alternatives, the fifth sequenceis codon optimized to reduce the total GC/AT ratio of the fifthsequence. In some alternatives, the fifth sequence is optimized by codonoptimization for expression in humans. In some alternatives, the codonoptimization and/or consensus sequence is generated by comparing thevariability of sequence and/or nucleobases utilized in a plurality ofrelated sequences. In some alternatives, the protein is a protein fortherapy. In some alternatives, the protein comprises an antibody or aportion thereof, which may be humanized. In some alternatives, thedouble mutant of dihydrofolate reductase comprises amino acid mutationsof L22F and F31S. In some alternatives, the gene delivery polynucleotideis a minicircle. In some alternatives, the introducing is performed byelectroporation. In some alternatives, the selecting is performed byincreasing selective pressure through the selective marker cassette. Insome alternatives, the selection reagent comprises an agent forselection. In some alternatives, the agent for selection ismethotrexate.

In some alternatives, wherein the first concentration range is at least50 nM-100 nM and the second concentration range is at least 75 to 150nM. In some alternatives, the first concentration range is at least 75nM-150 nM and the second concentration range is at least 112.5 nM to 225nM. In some alternatives, the first concentration range is at least 300nM-675 nM and the second concentration range is at least 450 nM to 1012nM. In some alternatives, the first round of selection comprisesexposing the cells to the selection agent for 2, 3, 4, 5, 6 or 7 daysbefore the second round of selection. In some alternatives, the secondround of selection comprises exposing the cells to the selection agentfor at least 2, 3, 4, 5, 6, or 7 days before isolation. In somealternatives, the precursor T cells are hematopoietic stem cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an overall schematic of the gene delivery minicircleproducer plasmid, MC_T3/FP-DHFRdm. The minicircle with T3 generation ofSleeping Beauty transposon comprises an EF1a promoter, a fusion offluorescent protein (FP; maxGFP, mCherry, or Blue Fluorescent protein(BFP)), Thosea asigna virus 2A peptide (T2A), and double mutant ofdihydrofolate reductase (DHFRdm) insensitive to methotrexate (MTX),positioned between inverted terminal repeats (ITRs, arrows).Recombination at attB/attP sites generates a minicircle while theremaining bacterial backbone is enzymatically degraded.

FIG. 2 shows a series of bar graphs that demonstrate the optimization ofthe transposon:transposase DNA ratio. H9 cells were nucleofected with 2μg of MC_T3/eGFP-T2A-DHFRdm DNA (transposon) and increasing amounts ofMC_SB100X (transposase) DNA (0.5, 1, 2, 4, 8 ug). Flow cytometry wasperformed at 24 hours (striped bars) and at 7 days (black bars) afternucleofection to assess transient and stable transfection efficiency.Numbers above the bars indicate integration efficiency, which iscalculated as percent of stable over transient GFP expression.

FIG. 3 shows a series of bar graphs, which demonstrate the effect of MTXconcentration during the selection process. Flow cytometric analysis ofH9 cell populations stably transfected with T3/GFP-T2A-DHFRdm transposonDNA grown in the presence of increasing concentrations of MTX (0, 50,100, and 200 nM) at 3 days (white bars), 5 days (horizontal stripes), 7days (vertical stripes), and 10 days (black bars) was performed. Panel Aof FIG. 3 shows the percent GFP+/PI− and Panel B of FIG. 3 shows themean GFP relative fluorescence units (RFU).

FIG. 4 shows a series of bar graphs that demonstrate the transgenepersistence after MTX withdrawal. As shown is the flow cytometricanalysis of H9 cell populations that were stably transfected withT3/GFP-T2A-DHFRdm transposon grown in media supplemented with differentconcentrations of MTX (50, 100, and 200 nM) for 2 weeks (black bars),after which MTX selection was withdrawn and data collected at differenttime points afterwards: 1 week (horizontal stripes), 2 weeks (verticalstripes), 3 weeks (checked bars), and 4 weeks (white bars). Panel A ofFIG. 4 shows the percent GFP+/PI−; Panel B of FIG. 4 shows the mean GFPrelative fluorescence units (RFU).

FIG. 5A shows the transposon copy number per human haploid genome.Genomic DNA was isolated from populations of H9 cells stably transfectedwith T3/GFP-T2A-DHFRdm transposon DNA before and after selection withdifferent concentrations of MTX (50, 100, and 200 nM). The averagetransposon copy number was determined by quantitative PCR. The “Goldstandard” was generated by the limiting dilution method. The “Sorted”population was created by sorting the original H9 population (8% ofintegrated transposon) to 100% GFP positive cells. The asterisk (*)above the bracketed bar graphs indicates the difference between 200 nMMTX and sorted population was significantly different according to aStudent's T-test (P=0.04).

FIG. 5B shows the distribution of transposon integration events. Sixtyclones were isolated by limited dilution method from an H9 populationthat was previously selected with 200 nM MTX to 100% cells withintegrated T3/GFP-T2A-DHFRdm transposon. Genomic DNA was isolated andtransposon copy number determined by relative RT-qPCR. Numbers wererounded to the nearest integer value (e.g., 0.5-1.5 was rounded to 1).N=60; mean±standard deviation=1.78±0.69. Probabilities of integrationevents and standard error were calculated from these data (inset table).

FIG. 6 shows a series of pie graphs representing the analysis of themultiplexing of transposons. As shown in Panels A-C are the flowcytometric analysis of H9 cell populations nucleofected with 3minicircles carrying transposons with different fluorescent proteins(FPs) (MC_T3/GFP-T2A-DHFRdm, MC_T3/BFP-T2A-DHFRdm,MC_T3/mCherry-T2A-DHFRdm), 2 μg each and 6 μg of MC_SB100X DNA atdifferent time points: (Panel A) 24 hours after transfection (transientexpression), (Panel B) 1 week (stable integration), and (Panel C) 1 weekof selection with 200 nM of MTX.

FIG. 7 shows the bar graph analysis of step selection of thedistribution of expression of single, double, and triple FPs. H9 cellpopulation stably transfected with three transposons was selected with200 nM MTX for a week and then was exposed to higher MTX concentrationsof 500 and 1000 nM.

FIG. 8 shows an example of the flow analysis for the stable expressionof transposon DNA with Sleeping Beauty in lymphocytes after MTXselection. Freshly thawed PBMC cells were electroporated with minicircleGFP (mcGFP) DNA (MC_T3/GFP-T2A-DHFRdm) and Sleeping Beauty transposaseDNA (MC_SB100X), then stimulated with Miltenyi Transact beads whichselectively activate T-cells by binding to CD3 and CD28. 1 week afterelectroporation, samples of the PBMC cells were selected using 25, 50and 100 nM MTX for 12 days (50 nM shown here). Panels A, B, and C showthe sequential selection for lymphocytes (A), single cells (B), and livecells (C). Shown in Panel D are the high levels of GFP expression inboth the CD8+ and CD8− populations. Note that for this donor, themajority of lymphocytes after stimulation are CD8+ T cells.

FIG. 9 shows histograms of the initial expression of transposon DNA withSleeping Beauty in lymphocytes. PBMC were transfected with either mcGFPDNA alone (10 ug), mcGFP (10 ug) and MC_SB100X DNA (5 ug) at amcGFP:MC_SB100X ratio of 2:1, mcGFP (10 ug) and MC_SB100X DNA (10 ug) ata mcGFP:MC_SB100X ratio of 1:1, a pMAXGFP (10 ug) control, or a no DNAcontrol. Shown in Panel A are the results for cells in which Transactbeads were not added, two days after transfection as an example of theinitial electroporation efficiency. Shown in Panel B are the results incells exposed to transact beads after five days. While by day 5 thelevels of mcGFP DNA decline to near control levels, the expression ofmcGFP in cells co-transfected with transposase remain elevated.

FIG. 10 shows the expression of GFP transposon DNA and the levels ofcell growth in transfected lymphocytes in the week before MTX addition.PBMC were transfected with either mcGFP DNA alone, mcGFP and MC_SB100XDNA at a mcGFP:MC_SB100X ratio of 2:1, mcGFP and MC_SB100X DNA at amcGFP:MC_SB100X ratio of 1:1, a pMAXGFP control (10 ug), or a no DNAcontrol. Panel A shows the decreasing levels of GFP expression from day2 to day 7. Panel B shows the level of live cells from day 0 to day 7 ofthe transfected cell samples which had been treated with MiltenyiTransact beads on d0. Panel C shows the level of live cells from day 0to day 7 of the transfected cell samples in the absence of Transactbeads. As shown, there is a slow growth of the cells transfected withmcGFP DNA in the presence of Miltenyi Transact beads.

FIG. 11 shows the stable expression of transposon DNA with SleepingBeauty in T-cells following 1 week of MTX selection. Shown are the flowcytometry scattergrams in which GFP production and proliferation ofT-cells modified to express GFP after transfection with transposon DNAand Sleeping Beauty transposase DNA were investigated. Panels A, B, E,and F show the scatter profiles to identify lymphocytes, while Panels C,D, G, and H show CD8 and GFP expression. Panels A-D show the flowcytometry analysis of cells treated with 100 nM MTX. Panels E-H showsthe flow cytometry analysis of cells that were not treated with MTX.Shown in Panels A, C, E and G, are samples transfected with mcGFP alone.Panels B, D, F and H show the flow cytometry results of cellstransfected with mcGFP and MC_SB100X (Sleeping Beauty transposase) DNAat 2:1. As demonstrated in Panel D, in T-cells (both CD8+ and CD8−)co-transfected with mcGFP and SB100X such that the GFP gene is stablyinserted into the cellular genome, about 95% of the cells stably expressGFP in the presence of MTX at 100 nM while only about 23% express GFP inthe absence of MTX.

FIG. 12 shows the proliferation and the GFP/CD8 expression intransposon-transfected lymphocytes after 14 days of MTX selection. Cellsamples were transfected with no DNA (control), mcGFP alone, mcGFP andMC_SB100X DNA at a mcGFP:MC_SB100X ratio of 2:1 ratio, or mcGFP andMC_SB100X DNA at a mcGFP:MC_SB100X ratio of 1:1. After 1 week, the cellswere selected using 0 nM MTX (control), 25 nM MTX, 50 nM MTX, or 100 nMMTX. The lymphocyte window, shown in the first, third, fifth and seventhcolumns, demonstrates the survival of only stably transfected cells inthe presence of higher concentrations of MTX. The live, singlelymphocytes were gated for GFP and CD8 detection in the second, fourth,sixth and eighth columns. For the cell samples transfected with mcGFPalone, GFP expression is lost over time (second column). However cellstransfected with both mcGFP and MC_SB100X stably express GFP both withMTX selection (>90%) and without MTX selection (˜20%) (columns four andsix). As shown in the samples transfected with mcGFP and MC_SB100X DNA,MTX was effective for selection at concentrations of 50 and 100 nM MTXand no significant difference was seen between the ratios 2:1 or 1:1.Note that the majority of lymphocytes are CD8+ T-cells.

FIG. 13 shows both the lymphocyte window and GFP/CD8 expression intransposon-transfected cells after 19 days of MTX selection. Cellsamples were transfected with no DNA (control), mcGFP alone, mcGFP andMC_SB100X DNA at a mcGFP:MC_SB100X ratio of 2:1 ratio, or mcGFP andMC_SB100X DNA at a mcGFP:MC_SB100X ratio of 1:1. The cells were selectedusing 0 nM MTX (control), 25 nM MTX, 50 nM MTX, or 100 nM MTX. Thelymphocyte window is shown in the first, third, fifth and seventhcolumns, showing the survival of only stably transfected cells in thepresence of MTX. The live, single lymphocytes were gated for GFP and CD8detection in the second, fourth, sixth and eighth columns. For the cellsamples transfected with mcGFP alone, GFP expression is lost over time(second column). However cells transfected with both mcGFP and MC_SB100Xstably express GFP both with MTX selection (>90%) and without MTXselection (˜20%) (columns four and six). As shown in the samplestransfected with mcGFP and MC_SB100X DNA, MTX was effective forselection at concentrations of 50 and 100 nM MTX, and slightly less for25 nM. The mcGFP:SB ratios 2:1 or 1:1 were similarly effective.

FIG. 14 shows the live cell counts of cells that stably expresstransposon DNA and undergoes MTX selection. Trypan blue cell counts weretaken at 7, 14, and 19 days post transfection. PBMC samples weretransfected with no DNA (control), mcGFP alone, mcGFP and MC_SB100X DNAat a mcGFP:MC_SB100X ratio of 2:1 ratio, or mcGFP and MC_SB100X DNA at amcGFP:MC_SB100X ratio of 1:1. The cells were selected on day 7 using 0nM MTX (control), 25 nM MTX, 50 nM MTX, or 100 nM MTX. Panel A, showsthe level of live cells in the absence of MTX. Panel B shows the levelsof live cells after exposure to 100 nM MTX. Panel C shows the levels oflive cells after exposure to 50 nM. Panel D shows the levels of livecells after exposure to 25 nM MTX. As MTX slows the growth of cells byinhibiting the metabolism of folic acid, only cells that weretransfected with both the mcGFP transposon co-expressing theMTX-resistance gene (DHFRdm) and the MC_SB100X plasmid encoding theSleeping Beauty Transposase were able to proliferate in the presence ofhigh MTX, due to stable expression of the integrated transposon DNA.

FIG. 15 shows an analysis of GFP expression by lymphocytes stablyexpressing GFP transposon DNA with Sleeping Beauty transposase under MTXselection. PBMC samples were transfected with mcGFP alone, mcGFP andMC_SB100X at a mcGFP:MC_SB100X ratio of 2:1, mcGFP and MC_SB100X at amcGFP:MC_SB100X ratio of 1:1, pMAXGFP (10 ug), and no DNA (control).Cells were exposed to MTX on day 7 after transfection, and GFPexpression was measured for live, single lymphocytes. Panel A shows thelevel of GFP expression on days 2, 5, 7, 14, and 19 in the absence ofMTX. Panel B shows the level of GFP expression from days 7, 14, and 19of lymphocytes transfected with mcGFP alone under MTX selection at MTXconcentrations of 0 nM, 25 nM, 50 nM and 100 nM. Panel C shows the GFPexpression of T-cells transfected with mcGFP and MC_SB100X at amcGFP:MC_SB100X ratio of 2:1 under MTX selection of 0 nM, 25 nM, 50 nMand 100 nM. Panel D shows the GFP expression of T-cells transfected withmcGFP and MC_SB100X at a mcGFP:MC_SB100X ratio of 1:1 under control ofMTX selection concentrations of 0 nM, 25 nM, 50 nM and 100 nM. As shown,the results from transfecting with mcGFP and MC_SB100X with a 2:1 and a1:1 ratio were similar, with approximately 75% GFP expression at 25 nMand approximately 90% GFP expression at 50 and 100 nM after 1 week ofMTX. Additionally, there was minimal difference in the GFP expressionbetween the treatment with 50 nM MTX and 100 nM MTX.

FIG. 16: Sleeping Beauty Transposons: minicircle constructs. As shown inthe figure are the schematics of several sleeping beauty constructsdesigned for several alternatives described herein.

FIG. 17: As shown are several scattergrams of cells transfected withSleeping Beauty transposons carrying a gene for expression of GFP. Asshown are the cells fourteen days after transfection. Cells wereelectroporated with SB100X or transposons carrying genes for GFP.

FIG. 18. Sleeping Beauty Transposons and MTX: GFP transposon. As shown,cells were transfected with different ratios of mcGFP plasmids and theSleeping Beauty transposon carrying a gene for expression of GFP (McGFP:SB at a 1:1 and 2:1 ratio). As shown, GFP expression was low with no MTXwas added after 18 days. With the Sleeping Beauty transposon, it isshown that there is an increase in GFP expression in the presence ofMTX.

FIG. 19. Sleeping Beauty Transposons: minicircle constructs. As shown inthe figure are the schematics of several sleeping beauty constructsdesigned for several alternatives described herein.

FIG. 20. Sleeping Beauty Transposons and MTX:GFP transposon—SB100X DNAand RNA. Cells were electroporated with SB100X (DNA or RNA) ortransposons carrying genes for GFP, CARs, or GFP/mCherry/BFP.

FIG. 21. Sleeping Beauty Transposons and MTX: GFP transposon—SB100X DNAand RNA. As shown in the figure are several scattergrams of the cellsthat are transfected with GFP gene carrying transposons. Several samplesof cells are transfected with DNA comprising a gene for GFP expression(2.5 ug and 5 ug), mcGFP only, and RNA (1 ug and 3 ug). The samples aresplit and grown under the influence of varying concentrations of MTX at0 uM, 50 uM and 100 uM.

FIG. 22. Sleeping Beauty Transposons and MTX: GFP transposon—SB100X DNAand RNA. Cells were transfected with Sleeping Beauty transposonscarrying a gene for GFP expression at different concentrations as seenin the top left panel. MTX was then added at day 7 after transfection.As shown, cells transfected with 50 ug to 100 ug can express GFP afterday 7 to day 14.

FIG. 23. GFP expressing DNA and RNA in the presence of MTX. As showncells transfected with mcGFP, GFP: SB, and GFP:SB RNA were grown andexposed to MTX seven days after transfection. As a control, cells weregrown to fourteen days without exposure to MTX (top left panel).

FIG. 24. Expression of GFP in cells transfected with GFP: SB. As shownin the left panel, cells were transfected with varying concentrations ofGFP: SB (2.5 ug, 5 ug) and exposed to different concentrations of MTX(50 uM and 100 uM). As shown, cells were able to express GFP in thepresence of MTX optimally at 50 uM MTX when they were transfected with 5ug of GFP: SB. This experiment was also performed using RNA, however,DNA has a higher efficiency for leading to expression of the protein.

FIG. 25. Sleeping Beauty Transposons: minicircle constructs. As shown inthe figure are the schematics of several sleeping beauty constructsdesigned for several alternatives described herein.

FIG. 26. Expression of CD19CAR. A Sleeping Beauty construct carrying agene for CD19CAR was constructed (SB: CD19CAR). Cells were transfectedwith either DNA (2.5 ug or 5 ug), or RNA (1 ug or 3 ug). As shown, cellsthat were transfected with DNA or RNA at both concentrations were ableto express the CD19CAR in the presence of 50 uM MTX. This was also shownfor cells that were transfected with the RNA at 1 ug in the presence of100 uM MTX.

FIG. 27. Expression of CD19CAR. A Sleeping Beauty construct carrying agene for CD19CAR was constructed (SB: CD19CAR). Cells were transfectedwith either DNA (2.5 ug or 5 ug), or RNA (1 ug or 3 ug). Cells weregrown and at day seven after transfection, were exposed to MTX. TheCD19CAR also included an EGFRt tag. As shown, detection of the tagcorrelates to the expression of the CD19CAR. After exposure to MTX,detection of the tag was seen in cells that were transfected with theDNA carrying the Sleeping Beauty construct carrying a gene for CAR19 aswell as the cells transfected with the RNA carrying the Sleeping Beautyconstruct carrying a gene for CAR19.

FIG. 28. Sleeping Beauty Transposons and MTX: CD19 CAR: CD8+ cellgrowth. Expression of CD19CAR. A Sleeping Beauty construct carrying agene for CD19CAR was constructed (SB: CD19CAR). Cells were transfectedwith either DNA (2.5 ug or 5 ug), or RNA (1 ug or 3 ug). Cells weregrown and at day seven after transfection, were exposed to MTX. Asshown, the CD8+ cells were able to grow when a lower concentration ofDNA was transfected. However, with RNA, it was seen that a higherconcentration led to better expression, but a lower concentration led tobetter initial growth of the cells.

FIG. 29. Sleeping Beauty Transposons: minicircle constructs. As shown inthe figure are the schematics of several sleeping beauty constructsdesigned for several alternatives described herein.

FIG. 30. Sleeping Beauty Transposons and MTX: Multiplex 3 FP's. Cellswere electroporated with DNA or mcFP and grown in the presence of MTX.Afterwards, cells were analyzed for expression of mCherry, BFP, and/orGFP as indicated by the scattergrams.

FIG. 31. Sleeping Beauty Transposons and MTX: Multiplex 3 FP's.

FIG. 32. Sleeping Beauty Transposons: minicircle constructs. As shown inthe figure are the schematics of several sleeping beauty constructsdesigned for several alternatives described herein.

FIG. 33. As shown, cells electroporated with DNA comprising SleepingBeauty transposons were subjected to different concentrations of MTX atthe second round of selection.

FIG. 34. Expression of Smarker proteins in cells electroporated with DNAcomprising Sleeping Beauty transposons in the presence of differentconcentrations of MTX (2, 100 nM, 250 nM, and 500 nM).

FIG. 35. Sleeping Beauty Transposons: minicircle constructs. As shown inthe figure are the schematics of several sleeping beauty constructsdesigned for several alternatives described herein.

DETAILED DESCRIPTION

The following definitions are provided to facilitate understanding ofthe embodiments or alternatives of the invention.

As used herein, “a” or “an” can mean one or more than one.

As used herein, the term “about” indicates that a value includes theinherent variation of error for the method being employed to determine avalue, or the variation that exists among experiments.

As used herein, “nucleic acid” or “nucleic acid molecule” refers topolynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), oligonucleotides, fragments generated by the polymerase chainreaction (PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acid molecules canbe composed of monomers that are naturally-occurring nucleotides (suchas DNA and RNA), or analogs of naturally-occurring nucleotides (e.g.,enantiomeric forms of naturally-occurring nucleotides), or a combinationof both. Modified nucleotides can have alterations in sugar moietiesand/or in pyrimidine or purine base moieties. Sugar modificationsinclude, for example, replacement of one or more hydroxyl groups withhalogens, alkyl groups, amines, and azido groups, or sugars can befunctionalized as ethers or esters. Moreover, the entire sugar moietycan be replaced with sterically and electronically similar structures,such as aza-sugars and carbocyclic sugar analogs. Examples ofmodifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called “peptide nucleic acids,” whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded. In some alternatives described herein, a gene deliverypolynucleotide for stable insertion of a nucleic acid into a gene isprovided. “Oligonucleotide” can be used interchangeable with nucleicacid and can refer to DNA or RNA, either double stranded or a singlestranded piece or DNA or RNA.

A “gene” is the molecular unit of heredity of a living organism,describing some stretches of deoxyribonucleic acids (DNA) andribonucleic acids (RNA) that code for a polypeptide or for an RNA chainthat has a function in the organism, and can be a locatable region inthe genome of an organism. In some alternatives described herein, a genedelivery polynucleotide for stable insertion of a nucleic acid into agene, wherein the nucleic acid for insertion is flanked by invertedterminal repeat gene sequences in the gene delivery polynucleotide andwherein the gene delivery polynucleotide is selectable, is provided.

A “chromosome,” is a packaged and organized chromatin, a complex ofmacromolecules found in cells, consisting of DNA, protein and RNA. Insome alternatives, a gene delivery polynucleotide for stable insertionof a nucleic acid into a gene, wherein the nucleic acid for insertion isflanked by inverted terminal repeat gene sequences in the gene deliverypolynucleotide and wherein the gene delivery polynucleotide isselectable, the gene delivery polynucleotide, is provided. In somealternatives, the nucleic acid is inserted into a gene of a chromosome.

A “promoter” is a nucleotide sequence that directs the transcription ofa structural gene. In some alternatives, a promoter is located in the 5′non-coding region of a gene, proximal to the transcriptional start siteof a structural gene. Sequence elements within promoters that functionin the initiation of transcription are often characterized by consensusnucleotide sequences. These promoter elements include RNA polymerasebinding sites, TATA sequences, CAAT sequences, differentiation-specificelements (DSEs; McGehee et al., Mol. Endocrinol. 7:551 (1993);incorporated by reference in its entirety), cyclic AMP response elements(CREs), serum response elements (SREs; Treisman, Seminars in CancerBiol. 1:47 (1990); incorporated by reference in its entirety),glucocorticoid response elements (GREs), and binding sites for othertranscription factors, such as CRE/ATF (O'Reilly et al., J. Biol. Chem.267:19938 (1992); incorporated by reference in its entirety), AP2 (Ye etal., J. Biol. Chem. 269:25728 (1994); incorporated by reference in itsentirety), SP1, cAMP response element binding protein (CREB; Loeken,Gene Expr. 3:253 (1993); incorporated by reference in its entirety) andoctamer factors (see, in general, Watson et al., eds., Molecular Biologyof the Gene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc.1987; incorporated by reference in its entirety)), and Lemaigre andRousseau, Biochem. J. 303:1 (1994); incorporated by reference in itsentirety). As used herein, a promoter can be constitutively active,repressible or inducible. If a promoter is an inducible promoter, thenthe rate of transcription increases in response to an inducing agent. Incontrast, the rate of transcription is not regulated by an inducingagent if the promoter is a constitutive promoter. Repressible promotersare also known. In some alternatives, a gene delivery polynucleotide isprovided. In some alternatives, the gene delivery polynucleotidecomprises a promoter sequence.

“Selectable marker cassette,” is a gene introduced into a vector or acell that confers a trait for artificial selection. A selectable markercassette can be a screenable marker to allow a researcher to distinguishbetween wanted and unwanted cells, or to enrich for a specific celltype. In some alternatives, a gene delivery polynucleotide is provided.In some alternatives, the gene delivery polynucleotide comprises aselectable marker cassette.

“Dihydrofolate reductase”, or DHFR, as described herein, is an enzymethat reduces dihydrofolic acid to tetrahydrofolic acid, using NADPH aselectron donor, which can be converted to the kinds of tetrahydrofolatecofactors used in 1-carbon transfer chemistry. In some alternativesdescribed herein, a gene delivery polynucleotide is provided. In somealternatives, the gene delivery polynucleotide comprises at least oneselectable marker cassette encoding for a double mutant of dihydrofolatereductase.

“Methotrexate” (MTX), as described herein, is an antimetabolite andantifolate drug. It acts by inhibiting the metabolism of folic acid. Insome alternatives, a method of generating engineered multiplexed T-cellsfor adoptive T-cell immunotherapy is provided. In the broadest sense,the method can comprise providing the gene delivery polynucleotide ofany of the alternatives described herein, introducing the gene deliverypolynucleotide into a T-cell, providing a vector encoding a SleepingBeauty transposase, introducing the vector encoding the Sleeping Beautytransposase into the T-cell, selecting the cells comprising the genedelivery polynucleotide, wherein the selecting comprises a first roundof selection and a second round of selection, wherein the first round ofselection comprises adding a selection reagent at a first concentrationrange and the second round of selection comprises adding the sameselection reagent at a second concentration range, wherein the secondconcentration range is greater than the first concentration range and,wherein the second concentration range is at least 1.5 fold higher thanthat of the first concentration range, and isolating the T-cellsexpressing a phenotype under this selective pressure. In somealternatives described herein, the selection reagent comprises an agentfor selection. In some alternatives, the selection reagent is MTX.

An “inverted repeat” or IR is a sequence of nucleotides followeddownstream by its reverse complement. Inverted repeats can have a numberof important biological functions. They can define the boundaries intransposons and indicate regions capable of self-complementary basepairing (regions within a single sequence which can base pair with eachother). These properties play an important role in genome instabilityand contribute to cellular evolution, genetic diversity and also tomutation and disease. In some alternatives, a gene deliverypolynucleotide is provided. In some alternatives, the gene deliverypolynucleotide comprises a first inverted terminal repeat gene sequenceand a second inverted terminal repeat gene sequence. In somealternatives, the gene delivery polynucleotide comprises a sleepingbeauty transposon positioned between two inverted repeat sequences.

Sleeping beauty transposase binds specific binding sites that arelocated on the IR of the Sleeping beauty transposon. The sequence of IR(Inverted repeat) is as follows

(SEQ ID NO: 1) cagttgaagtcggaagtttacatacacttaagttggagtcattaaaactcgtttttcaactacTccacaaatttcttgttaacaaacaatagttttggcaagtcagttaggacatctactttgtgcatgacacaagtcatttttccaacaattgtttacagacagattatttcacttataattcactgtatcacaattccagtgggtcagaagtttacatacactaagttgactgtgcctttaaacagcttggaaaattccagaaaatgatgtcatggctttagaagcttctgatagactaattgacatcatttgagtcaattggaggtgtacctg tggatgtatttcaagg

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides.”

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein can also comprise non-peptide components, such ascarbohydrate groups. Carbohydrates and other non-peptide substituentscan be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but can be presentnonetheless. In some alternatives, a gene delivery polynucleotide forstable insertion of a nucleic acid into a gene, wherein the nucleic acidfor insertion is flanked by inverted terminal repeat gene sequences inthe gene delivery polynucleotide and wherein the gene deliverypolynucleotide is selectable, the gene delivery polynucleotide, isprovided. In some alternatives, the gene delivery polynucleotide furthercomprises a sequence for at least one protein.

An “antibody” as described herein refers to a large Y-shape proteinproduced by plasma cells that is used by the immune system to identifyand neutralize foreign objects such as bacteria and viruses. Theantibody protein can comprise four polypeptide chains; two identicalheavy chains and two identical light chains connected by disulfidebonds. Each chain is composed of structural domains calledimmunoglobulin domains. These domains can contain about 70-110 aminoacids and are classified into different categories according to theirsize and function. In some alternatives, a gene delivery polynucleotidefor stable insertion of a nucleic acid into a gene, wherein the nucleicacid for insertion is flanked by inverted terminal repeat gene sequencesin the gene delivery polynucleotide and wherein the gene deliverypolynucleotide is selectable, the gene delivery polynucleotide, isprovided. In some alternatives, the gene delivery polynucleotide furthercomprises a sequence for at least one protein. In some alternatives, thegene delivery polynucleotide can comprise a sequence for an antibody ora portion thereof, which may be humanized.

A “chimeric antigen receptor” (CARs), also known as chimeric T-cellreceptors, refers to artificial T-cell receptors that are engineeredreceptors, which graft an arbitrary specificity onto an immune effectorcell. These receptors can be used to graft the specificity of amonoclonal antibody onto a T-cell, for example; with transfer of theircoding sequence facilitated by retroviral vectors. The structure of theCAR can comprise single-chain variable fragments (scFv) derived frommonoclonal antibodies, fused to CD3-zeta transmembrane and endodomain.Such molecules result in the transmission of a zeta signal in responseto recognition by the scFv of its target. Some alternatives utilize agene delivery polynucleotide for stable insertion of a nucleic acid intoa gene, wherein the nucleic acid for insertion is flanked by invertedterminal repeat gene sequences in the gene delivery polynucleotide, andwherein the gene delivery polynucleotide is selectable. In somealternatives, the gene delivery polynucleotide further comprises asequence for at least one protein. In some alternatives, the protein isa chimeric antigen receptor. Chimeric receptor can also be referred toas artificial T cell receptors, chimeric T cell receptors, chimericimmunoreceptors, and chimeric antigen receptors (CARs). These CARs areengineered receptors that can graft an arbitrary specificity onto animmune receptor cell. Chimeric antigen receptors or “CARs” areconsidered by some investigators in some contexts to include theantibody or antibody fragment, spacer, signaling domain, andtransmembrane region. However, due to the surprising effects ofmodifying the different components or domains of the CAR, such as theepitope binding region (for example, antibody fragment, scFv, or portionthereof), spacer, transmembrane domain, and/or signaling domain), thecomponents of the CAR are described herein in some contexts to includethese features as independent elements. The variation of the differentelements of the CAR can, for example, lead to stronger binding affinityfor a specific epitope.

Artificial T-cell receptors, or CARs can be used as a therapy for canceror viral infection using a technique called adoptive cell transfer.T-cells are removed from a patient and modified so that they expressreceptors specific for a molecule displayed on a cancer cell or virus,or virus-infected cell. The genetically engineered T-cells, which canthen recognize and kill the cancer cells or the virus infected cells orpromote clearance of the virus, are reintroduced into the patient. Insome alternatives, the gene delivery polynucleotide can comprise asequence for a chimeric antigen receptor. In some alternatives, a methodof generating engineered multiplexed T-cells for adoptive T-cellimmunotherapy is provided. In the broadest sense the method can compriseproviding the gene delivery polynucleotide of any one of thealternatives described herein, introducing the gene deliverypolynucleotide into a T-cell, providing a vector encoding a SleepingBeauty transposase, introducing the vector encoding the Sleeping Beautytransposase into the T-cell, selecting the cells comprising the genedelivery polynucleotide, wherein selecting comprises a first round ofselection and a second round of selection, wherein the first round ofselection comprises adding a selection reagent at a first concentrationrange and the second round of selection comprises adding the selectionreagent at a second concentration range, and wherein the secondconcentration range is at least 1.5 fold higher than that of the firstconcentration range and isolating the T-cells expressing a phenotypeunder selective pressure. In some alternatives, the selection reagent isMTX.

T-cell co-stimulation is desired for development of an effective immuneresponse and this event occurs during the activation of lymphocytes. Aco-stimulatory signal, is antigen non-specific and is provided by theinteraction between co-stimulatory molecules expressed on the membraneof the antigen bearing cell and the T-cell. Co-stimulatory molecules caninclude but are not limited to CD28, CD80, and CD86. In somealternatives, a method for generating engineered multiplexed T-cell foradoptive T-cell immunotherapy is provided. In some alternatives, theT-cell is a chimeric antigen receptor bearing T-cell. In somealternatives, the chimeric antigen receptor bearing T-cell is engineeredto express co-stimulatory ligands. In some alternatives, methods areprovided for treating, inhibiting, or ameliorating cancer or a viralinfection in a subject. In the broadest sense the method can compriseadministering to the subject a T-cell of any of the alternativesdescribed herein. Preferably, genetically engineered T cells are used totreat, inhibit, or ameliorate a cancer or a viral disease, wherein thegenetically engineered T cells are obtained by preferentialamplification of T cells that are transformed to express multipletransgenes encoding receptors or chimeric receptors specific for amolecule presented by a virus or a cancer cell and selection pressure onthe transformed T cells is applied in a two-stage MTX selection,utilizing increasing concentrations of MTX. In some of thesealternatives, the subject is an animal, such as domestic livestock or acompanion animal and on other alternatives, the subject is a human. Insome of these alternatives, the chimeric antigen bearing T-cell isengineered to express a co-stimulatory molecule. In some alternatives,the gene delivery polynucleotide comprises a sequence for at least oneco-stimulatory molecule. In some alternatives, the gene deliverypolynucleotide is circular. In some alternatives, the gene deliverypolynucleotide is at least 1 kB to 6 kB. In some alternatives, the genedelivery polynucleotide is a minicircle.

“T cell precursors” as described herein refers to lymphoid precursorcells that can migrate to the thymus and become T cell precursors, whichdo not express a T cell receptor. All T cells originate fromhematopoietic stem cells in the bone marrow. Hematopoietic progenitors(lymphoid progenitor cells) from hematopoietic stem cells populate thethymus and expand by cell division to generate a large population ofimmature thymocytes. The earliest thymocytes express neither CD4 norCD8, and are therefore classed as double-negative (CD4⁻CD8⁻) cells. Asthey progress through their development, they become double-positivethymocytes (CD4⁺CD8⁺), and finally mature to single-positive (CD4⁺CD8⁻or CD4⁻CD8⁺) thymocytes that are then released from the thymus toperipheral tissues.

About 98% of thymocytes die during the development processes in thethymus by failing either positive selection or negative selection,whereas the other 2% survive and leave the thymus to become matureimmunocompetent T cells.

The double negative (DN) stage of the precursor T cell is focused onproducing a functional β-chain whereas the double positive (DP) stage isfocused on producing a functional α-chain, ultimately producing afunctional αβ T cell receptor. As the developing thymocyte progressesthrough the four DN stages (DN1, DN2, DN3, and DN4), the T cellexpresses an invariant α-chain but rearranges the β-chain locus. If therearranged β-chain successfully pairs with the invariant α-chain,signals are produced which cease rearrangement of the β-chain (andsilence the alternate allele) and result in proliferation of the cell.Although these signals require this pre-TCR at the cell surface, theyare dependent on ligand binding to the pre-TCR. These thymocytes willthen express both CD4 and CD8 and progresses to the double positive (DP)stage where selection of the α-chain takes place. If a rearrangedβ-chain does not lead to any signaling (e.g. as a result of an inabilityto pair with the invariant α-chain), the cell may die by neglect (lackof signaling).

“Hematopoietic stem cells” or “HSC” as described herein, are precursorcells that can give rise to myeloid cells such as, for example,macrophages, monocytes, macrophages, neutrophils, basophils,eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells andlymphoid lineages (such as, for example, T-cells, B-cells, NK-cells).HSCs have a heterogeneous population in which three classes of stemcells exist, which are distinguished by their ratio of lymphoid tomyeloid progeny in the blood (L/M).

In some alternatives, a method of generating engineered multiplexedT-cells for adoptive T-cell immunotherapy is provided, wherein themethod comprises providing a gene delivery polynucleotide, introducingthe gene delivery polynucleotide into a T-cell, providing a vectorencoding a Sleeping Beauty transposase, introducing the vector encodingthe Sleeping Beauty transposase into the T-cell, selecting the cellscomprising the gene delivery polynucleotide wherein selecting comprisesa first round of selection and a second round of selection, wherein thefirst round of selection comprises adding a selection reagent at a firstconcentration range and the second round of selection comprises addingthe selection reagent at a second concentration range, wherein thesecond concentration range is higher than the first concentration rangeand, wherein the second concentration range is at least 1.5 fold higherthan that of the first concentration range and isolating the T-cellsexpressing a phenotype under selective pressure. In some alternatives,the gene delivery polynucleotide comprises a first sequence, wherein thefirst sequence comprises a first inverted terminal repeat gene sequence,a second sequence, wherein the second sequence comprises a secondinverted terminal repeat gene sequence, a third sequence, wherein thethird sequence comprises a promoter region sequence, a fourth sequence,wherein the fourth sequence comprises at least one gene encoding aprotein, and wherein the fourth sequence is optimized, a fifth sequence,wherein the fifth sequence comprises at least one selectable markercassette encoding a double mutant of dihydrofolate reductase, whereinthe double mutant of dihydrofolate reductase has a 15,000 fold or about15,000 fold reduced affinity for methotrexate, wherein the methotrexatecan be used as a selection mechanism to selectively amplify cellstransduced with the gene delivery polynucleotide and wherein the fifthsequence is optimized, a sixth sequence, wherein the sixth sequencecomprises a first attachment site (attP) and a seventh sequence, whereinthe seventh sequence comprises a second attachment site (attB) whereineach of the first sequence, second sequence, third sequence, fourthsequence, fifth sequence, sixth sequence, and seventh sequence have a 5′terminus and a 3′ terminus, and wherein the 3′ terminus of the firstsequence comprising the first inverted terminal repeat gene sequence isadjacent to the 5′ terminus of the third sequence, the 3′ terminus ofthe third sequence is adjacent to the 5′ terminus of the fourthsequence, the 3′ terminus of the fourth sequence is adjacent to the 5′terminus of the fifth sequence and the 3′ terminus of the fifth sequenceis adjacent to the 5′ terminus of the second sequence comprising asecond inverted terminal repeat. In some alternatives, the gene encodingthe double mutant of human dihydrofolate reductase comprises the DNAsequence: ATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACTTCCCCTGGCCACCGCTCAGGAATGAATCCAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAAGGAGCTCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAGAATGATTAA (SEQ ID NO: 2). In some alternatives, the doublemutant of human dihydrofolate reductase comprises the protein sequence:MVGSLNCIVA VSQNMGIGKN GDFPWPPLRN ESRYFQRMTT TSSVEGKQNL VIMGKKTWFSIPEKNRPLKG RINLVLSREL KEPPQGAHFL SRSLDDALKL TEQPELANKV DMVWIVGGSSVYKEAMNHPG HLKLFVTRIM QDFESDTFFP EIDLEKYKLL PEYPGVLSDV QEEKGIKYKFEVYEKND (SEQ ID NO: 3). In some alternatives, the gene deliverypolynucleotide is circular. In some alternatives, the gene deliverypolynucleotide is at least 1 kB to 5 kB. In some alternatives, the genedelivery polynucleotide is a minicircle. In some alternatives, thepromoter region comprises an EF1 promoter sequence. In somealternatives, the fourth sequence comprises one, two, three, four, orfive genes that encode proteins. In some alternatives, the fourthsequence is codon optimized to reduce the total GC/AT ratio of thefourth sequence. In some alternatives, the fourth sequence is optimizedby codon optimization for expression in humans. In some alternatives,the fourth sequence is a consensus sequence generated from a pluralityof nucleic acids that encode a plurality of related proteins. In somealternatives, the fourth sequence is a consensus sequence generated froma plurality of nucleic acids that encode a plurality of relatedproteins, such as a plurality of antibody binding domains, which arespecific for the same epitope. In some alternatives, the plurality ofrelated proteins comprise a plurality of antibody binding domains,wherein the plurality of antibody binding domains are specific for thesame epitope. In some alternatives, the fifth sequence is codonoptimized to reduce the total GC/AT ratio of the fifth sequence. In somealternatives, the fifth sequence is optimized by codon optimization forexpression in humans. In some alternatives, the protein is a protein fortherapy. In some alternatives, the codon optimization and/or consensussequence is generated by comparing the variability of sequence and/ornucleobases utilized in a plurality of related sequences. In somealternatives, the protein comprises an antibody or a portion thereof,which may be humanized. In some alternatives, the double mutant ofdihydrofolate reductase comprises amino acid mutations of L22F and F31S.In some alternatives, the double mutant of dihydrofolate reductasecomprises amino acid mutations of L22F and F31S. In some alternatives,the introducing is performed by electroporation. In some alternatives,the selecting is performed by increasing selective pressure through theselective marker cassette. In some alternatives, the selection reagentcomprises an agent for selection. In some alternatives, the agent forselection is methotrexate. In some alternatives, the first concentrationrange is at least 50 nM-100 nM and the second concentration range is atleast 75 to 150 nM. In some alternatives, the first concentration is 50nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100 nM or any concentration that isbetween a range of concentrations defined by any two of theaforementioned concentrations, and the second concentration range is 75nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, or 150 nM orany concentration that is between a range of concentrations defined byany two of the aforementioned concentrations. In some alternatives, thefirst concentration range is at least 75 nM-150 nM and the secondconcentration range is at least 112.5 nM to 225 nM. In somealternatives, the first concentration is 75 nM, 85 nM, 95 nM, 105 nM,115 nM, 125 nM, 135 nM, 145 nM, or 150 nM or any concentration that isbetween a range of concentrations defined by any two of theaforementioned concentrations, and the second concentration range is 112nM, 122 nM, 132 nM, 142 nM, 152 nM, 162 nM, 172 nM, 182 nM, 192 nM, 202nM, 212 nM, or 225 nM or any concentration that is between a range ofconcentrations defined by any two of the aforementioned concentrations.In some alternatives, the first concentration range is at least 300nM-675 nM and the first concentration range is at least 450 nM to 1012nM. In some alternatives, the first concentration is 300 nM, 350 nM, 400nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, or 675 nM or anyconcentration that is between a range of concentrations defined by anytwo of the aforementioned concentrations, and the second concentrationrange is 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM,850 nM, 900 nM, 1000 nM, or 1012 nM or any concentration that is betweena range of concentrations defined by any two of the aforementionedconcentrations. In some alternatives, the first round of selectioncomprises exposing the T-cells to the selection agent for 2, 3, 4, 5, 6or 7 days before the second round of selection. In some alternatives,the second round of selection comprises exposing the T-cells to theselection agent for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or14 days or any time that is between a range of times defined by any twoof the aforementioned time points before isolation. In somealternatives, the T cells comprise precursor T cells. In somealternatives, the precursor T cells are hematopoietic stem cells.

In some alternatives, a method of generating engineered cells foradoptive T-cell immunotherapy comprising, providing a gene deliverypolynucleotide, introducing the gene delivery polynucleotide into aprecursor T cell, providing a vector encoding a Sleeping Beautytransposase, introducing the vector encoding the Sleeping Beautytransposase into the precursor T cell, selecting the precursor T cellscomprising the gene delivery polynucleotide; wherein selecting comprisesa first round of selection and a second round of selection, wherein thefirst round of selection comprises adding a selection reagent at a firstconcentration range and the second round of selection comprises addingthe selection reagent at a second concentration range, wherein thesecond concentration range is higher than the first concentration rangeand, wherein the second concentration range is at least 1.5 fold higherthan that of the first concentration range and isolating the precursorT-cells expressing a phenotype under selective pressure. In somealternatives, the gene delivery polynucleotide is for stable insertionof a nucleic acid into an oligonucleotide wherein the nucleic acid forinsertion is flanked by inverted terminal repeat gene sequences in thegene delivery polynucleotide and wherein the gene deliverypolynucleotide is selectable, wherein the gene delivery polynucleotidecomprises a first sequence, wherein the first sequence comprises a firstinverted terminal repeat gene sequence, a second sequence, wherein thesecond sequence comprises a second inverted terminal repeat genesequence, a third sequence, wherein the third sequence comprises apromoter region sequence, a fourth sequence, wherein the fourth sequencecomprises at least one gene, wherein the at least one gene encodes aprotein or encodes a sequence for mRNA transcription, and wherein thefourth sequence is optimized, a fifth sequence, wherein the fifthsequence comprises at least one selectable marker cassette encoding adouble mutant of dihydrofolate reductase, wherein the double mutant ofdihydrofolate reductase has a 15,000 fold or about 15,000 fold reducedaffinity for methotrexate, wherein the methotrexate can be used toselect for cells transduced with the gene delivery polynucleotide, toenhance the ratio of cells expressing the at least one gene and whereinthe fifth sequence is optimized, a sixth sequence, wherein the sixthsequence comprises a first attachment site (attP) and a seventhsequence, wherein the seventh sequence comprises a second attachmentsite (attB); wherein each of the first sequence, second sequence, thirdsequence, fourth sequence, fifth sequence, sixth sequence, and seventhsequence have a 5′ terminus and a 3′ terminus, and wherein the 3′terminus of the first sequence comprising the first inverted terminalrepeat gene sequence is adjacent to the 5′ terminus of the thirdsequence, the 3′ terminus of the third sequence is adjacent to the 5′terminus of the fourth sequence, the 3′ terminus of the fourth sequenceis adjacent to the 5′ terminus of the fifth sequence and the 3′ terminusof the fifth sequence is adjacent to the 5′ terminus of the secondsequence comprising a second inverted terminal repeat. In somealternatives, the gene delivery polynucleotide is circular. In somealternatives, the gene delivery polynucleotide is at least 1 kB to 5 kB.In some alternatives, the promoter region comprises an EF1 promotersequence. In some alternatives, the fourth sequence comprises one, two,three, four, or five genes that encode proteins. In some alternatives,the fourth sequence is codon optimized to reduce the total GC/AT ratioof the fourth sequence. In some alternatives, the fourth sequence isoptimized by codon optimization for expression in humans. In somealternatives, the fourth sequence is a consensus sequence generated froma plurality of nucleic acids that encode a plurality of relatedproteins. In some alternatives, the fourth sequence is a consensussequence generated from a plurality of nucleic acids that encode aplurality of related proteins, such as a plurality of antibody bindingdomains, which are specific for the same epitope. In some alternatives,the plurality of related proteins comprise a plurality of antibodybinding domains, wherein the plurality of antibody binding domains arespecific for the same epitope. In some alternatives, the fifth sequenceis codon optimized to reduce the total GC/AT ratio of the fifthsequence. In some alternatives, the fifth sequence is optimized by codonoptimization for expression in humans. In some alternatives, the codonoptimization and/or consensus sequence is generated by comparing thevariability of sequence and/or nucleobases utilized in a plurality ofrelated sequences. In some alternatives, the protein is a protein fortherapy. In some alternatives, the protein comprises an antibody or aportion thereof, which may be humanized. In some alternatives, thedouble mutant of dihydrofolate reductase comprises amino acid mutationsof L22F and F31S. In some alternatives, the gene delivery polynucleotideis a minicircle. In some alternatives, the introducing is performed byelectroporation. In some alternatives, the selecting is performed byincreasing selective pressure through the selective marker cassette. Insome alternatives, the selection reagent comprises an agent forselection. In some alternatives, the agent for selection ismethotrexate. In some alternatives, the first concentration range is atleast 50 nM-100 nM and the second concentration range is at least 75 to150 nM. In some alternatives, the first concentration range is at least75 nM-150 nM and the second concentration range is at least 112.5 nM to225 nM. In some alternatives, the first concentration range is at least300 nM-675 nM and the first concentration range is at least 450 nM to1012 nM. In some alternatives, the first round of selection comprisesexposing the T-cells to the selection agent for 2, 3, 4, 5, 6 or 7 daysbefore the second round of selection. In some alternatives, the secondround of selection comprises exposing the T-cells to the selection agentfor at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or anytime that is between a range of times defined by any two of theaforementioned time points before isolation. In some alternatives, the Tcell precursor is a hematopoietic stem cell.

In some alternatives, a method of increasing protein production in aprecursor T-cell is provided wherein the method comprises providing apolynucleotide, introducing the polynucleotide into a cell, providing avector encoding a Sleeping Beauty transposase; introducing the vectorencoding the Sleeping Beauty transposase into the precursor T-cell,selecting the precursor T cells comprising the gene deliverypolynucleotide, wherein selecting comprises a first round of selectionand a second round of selection, wherein the first round of selectioncomprises adding a selection reagent at a first concentration range andthe second round of selection comprises adding the selection reagent ata second concentration range, wherein the second concentration range ishigher than the first concentration range and, wherein the secondconcentration range is at least 1.5 fold higher than that of the firstconcentration range and isolating the precursor T cells expressing aphenotype under selective pressure. In some alternatives, the genedelivery polynucleotide is for stable insertion of a nucleic acid intoan oligonucleotide wherein the nucleic acid for insertion is flanked byinverted terminal repeat gene sequences in the gene deliverypolynucleotide and wherein the gene delivery polynucleotide isselectable, wherein the gene delivery polynucleotide comprises a firstsequence, wherein the first sequence comprises a first inverted terminalrepeat gene sequence, a second sequence, wherein the second sequencecomprises a second inverted terminal repeat gene sequence, a thirdsequence, wherein the third sequence comprises a promoter regionsequence, a fourth sequence, wherein the fourth sequence comprises atleast one gene, wherein the at least one gene encodes a protein orencodes a sequence for mRNA transcription, and wherein the fourthsequence is optimized, a fifth sequence, wherein the fifth sequencecomprises at least one selectable marker cassette encoding a doublemutant of dihydrofolate reductase, wherein the double mutant ofdihydrofolate reductase has a 15,000 fold or about 15,000 fold reducedaffinity for methotrexate, wherein the methotrexate can be used toselect for cells transduced with the gene delivery polynucleotide, toenhance the ratio of cells expressing the at least one gene and whereinthe fifth sequence is optimized, a sixth sequence, wherein the sixthsequence comprises a first attachment site (attP) and a seventhsequence, wherein the seventh sequence comprises a second attachmentsite (attB); wherein each of the first sequence, second sequence, thirdsequence, fourth sequence, fifth sequence, sixth sequence, and seventhsequence have a 5′ terminus and a 3′ terminus, and wherein the 3′terminus of the first sequence comprising the first inverted terminalrepeat gene sequence is adjacent to the 5′ terminus of the thirdsequence, the 3′ terminus of the third sequence is adjacent to the 5′terminus of the fourth sequence, the 3′ terminus of the fourth sequenceis adjacent to the 5′ terminus of the fifth sequence and the 3′ terminusof the fifth sequence is adjacent to the 5′ terminus of the secondsequence comprising a second inverted terminal repeat. In somealternatives, the gene delivery polynucleotide is circular. In somealternatives, the gene delivery polynucleotide is at least 1 kB to 5 kB.In some alternatives, the promoter region comprises an EF1 promotersequence. In some alternatives, the fourth sequence comprises one, two,three, four, or five genes that encode proteins. In some alternatives,the fourth sequence is codon optimized to reduce the total GC/AT ratioof the fourth sequence. In some alternatives, the fourth sequence isoptimized by codon optimization for expression in humans. In somealternatives, the fourth sequence is a consensus sequence generated froma plurality of nucleic acids that encode a plurality of relatedproteins. In some alternatives, the fourth sequence is a consensussequence generated from a plurality of nucleic acids that encode aplurality of related proteins, such as a plurality of antibody bindingdomains, which are specific for the same epitope. In some alternatives,the plurality of related proteins comprise a plurality of antibodybinding domains, wherein the plurality of antibody binding domains arespecific for the same epitope. In some alternatives, the fifth sequenceis codon optimized to reduce the total GC/AT ratio of the fifthsequence. In some alternatives, the fifth sequence is optimized by codonoptimization for expression in humans. In some alternatives, the codonoptimization and/or consensus sequence is generated by comparing thevariability of sequence and/or nucleobases utilized in a plurality ofrelated sequences. In some alternatives, the protein is a protein fortherapy. In some alternatives, the protein comprises an antibody or aportion thereof, which may be humanized. In some alternatives, thedouble mutant of dihydrofolate reductase comprises amino acid mutationsof L22F and F31S. In some alternatives, the gene delivery polynucleotideis a minicircle. In some alternatives, the introducing is performed byelectroporation. In some alternatives, the selecting is performed byincreasing selective pressure through the selective marker cassette. Insome alternatives, the selection reagent comprises an agent forselection. In some alternatives, the agent for selection ismethotrexate.

In some alternatives, wherein the first concentration range is at least50 nM-100 nM and the second concentration range is at least 75 to 150nM. In some alternatives, the first concentration range is at least 75nM-150 nM and the second concentration range is at least 112.5 nM to 225nM. In some alternatives, the first concentration range is at least 300nM-675 nM and the second concentration range is at least 450 nM to 1012nM. In some alternatives, the first round of selection comprisesexposing the cells to the selection agent for 2, 3, 4, 5, 6 or 7 daysbefore the second round of selection. In some alternatives, the secondround of selection comprises exposing the cells to the selection agentfor at least 2, 3, 4, 5, 6, or 7 days before isolation. In somealternatives, the precursor T cells are hematopoietic stem cells.

A peptide or polypeptide encoded by a non-host DNA molecule is a“heterologous” peptide or polypeptide.

An “integrated genetic element” is a segment of DNA that has beenincorporated into a chromosome of a host cell after that element isintroduced into the cell through human manipulation. Within the presentalternatives, integrated genetic elements can be derived fromminicircles that are introduced into the cells by electroporation orother techniques. Integrated genetic elements are passed from theoriginal host cell to its progeny. In some alternatives, an integratedgenetic element is incorporated into a chromosome of a host cell by agene delivery polynucleotide is circular. In some alternatives, the genedelivery polynucleotide is at least 1 kB to 6 kB. In some alternatives,the gene delivery polynucleotide is a minicircle.

A “cloning vector” or vector is a nucleic acid molecule, such as aminicircle, plasmid, cosmid, plastome, or bacteriophage that has thecapability of replicating autonomously in a host cell. Cloning vectorstypically contain one or a small number of restriction endonucleaserecognition sites that allow insertion of a nucleic acid molecule in adeterminable fashion without loss of an essential biological function ofthe vector, as well as nucleotide sequences encoding a marker gene thatis suitable for use in the identification and selection of cellstransduced with the cloning vector. Marker genes typically include genesthat provide tetracycline resistance or ampicillin resistance but insome alternatives can include a methotrexate resistance gene.

An “expression vector” is a nucleic acid molecule encoding a gene thatis expressed in a host cell. Typically, an expression vector comprises atranscription promoter, a gene, and a transcription terminator. Geneexpression is usually placed under the control of a promoter, and such agene is said to be “operably linked to” the promoter. Similarly, aregulatory element and a core promoter are operably linked if theregulatory element modulates the activity of the core promoter. In somealternatives, an expression vector is provided. In some alternatives,the expression vector encodes a transposase. In some alternatives, thetransposase is a Sleeping Beauty transposase. In some alternatives,expression vector is circular. In some alternatives, the expressionvector is at least 1 kB to 6 kB. In some alternatives, the expressionvector is a minicircle.

“Minicircles,” as described herein, are small circular plasmidderivatives that have been freed from all prokaryotic vector parts.Minicircles can serve as an expression vector, where they have beenapplied as transgene carriers for the genetic modification of mammaliancells, with the advantage that, since they contain no bacterial DNAsequences, they are less likely to be perceived as foreign anddestroyed. As such, typical transgene delivery methods involve plasmids,which contain foreign DNA. The smaller size of minicircles also extendstheir cloning capacity and facilitates their delivery into cells.Without being limiting, the preparation of minicircles can follow atwo-step procedure, which can involve production of a parental plasmid(bacterial plasmid with eukaryotic inserts) in E. coli and induction ofa site-specific recombinase at the end of this process but still inbacteria. These steps can be followed by the excision of prokaryoticvector parts via two recombinase-target sequences at both ends of theinsert and recovery of the resulting minicircle (vehicle for the highlyefficient modification of the recipient cell) and the miniplasmid bycapillary gel electrophoresis (CGE).

The purified minicircle can be transferred into the recipient cell bytransfection, by electroporation, or by other methods known to thoseskilled in the art. Conventional minicircles can lack an origin ofreplication, so they cannot replicate within the target cells and theencoded genes will disappear as the cell divides (which can be either anadvantage or disadvantage depending on whether the application demandspersistent or transient expression). Some alternatives utilize a genedelivery polynucleotide for stable insertion of a nucleic acid into agene, wherein the nucleic acid for insertion is flanked by invertedterminal repeat gene sequences in the gene delivery polynucleotide, andwherein the gene delivery polynucleotide is selectable. In somealternatives, the gene delivery polynucleotide is a minicircle.

As used herein, “nucleofection”, refers to a transfection method ofexogenous nucleic acid(s) into a host cell and is performed byelectroporation. In some alternatives, a method of generating engineeredmultiplexed T-cells for adoptive T-cell immunotherapy is provided. Inthe broadest sense the method can comprise providing the gene deliverypolynucleotide of any of the alternatives described herein, introducingthe gene delivery polynucleotide into a T-cell, selecting the cellscomprising the gene delivery polynucleotide, wherein selecting comprisesa first round of selection and a second round of selection, wherein thefirst round of selection comprises adding a selection reagent at a firstconcentration range and the second round of selection comprises addingthe selection reagent at a second concentration range, wherein thesecond concentration range is higher than the first concentration rangeand, wherein the second concentration range is at least 1.5 fold higherthan that of the first concentration range and isolating the T-cellsexpressing a phenotype under selective pressure. In some alternatives,the selection reagent is MTX. In some alternatives, introducing the genedelivery polynucleotide into a T-cell can be performed byelectroporation.

“Host cell” as described herein, is a cell that contains one or morenucleases, for example endonucleases, end-processing enzymes, and/orendonuclease/end-processing enzyme fusion proteins encompassed by thepresent alternatives or a vector encoding the same that supports thereplication, and/or transcription or transcription and translation(expression) of one or more nucleases, for example endonucleases,end-processing enzymes, and/or endonuclease/end-processing enzyme fusionproteins. In some alternatives, host cells for use in the presentalternatives can be eukaryotic cells. Host cells of the immune systemcan include T-cells. In some alternatives, a method of generatingengineered multiplexed T-cells for adoptive T-cell immunotherapy isprovided. In some alternatives, the method can comprise providing thegene delivery polynucleotide of any one of the alternatives describedherein, introducing the gene delivery polynucleotide into a T-cell,providing a vector encoding a Sleeping Beauty transposase, introducingthe vector encoding the Sleeping Beauty transposase into the T-cell,selecting the cells comprising the gene delivery polynucleotide, whereinthe selecting comprises a first round of selection and a second round ofselection, wherein the first round of selection comprises adding aselection reagent at a first concentration range and the second round ofselection comprises adding the selection reagent at a secondconcentration range, wherein the second concentration range is higherthan the first concentration range and, wherein the second concentrationrange is at least 1.5 fold higher than that of the first concentrationrange and isolating the T-cells expressing a phenotype under selectivepressure. In some alternatives, the selection reagent is MTX.

As described herein, “transposable element” (TE), transposon orretrotransposon, can be referred to as a DNA sequence that can changeits position within the genome, sometimes creating or reversingmutations and altering the cell's genome size. Transposition oftenresults in duplication of the TE. TEs can make up a large fraction ofthe C-value of eukaryotic cells. “C-values,” as described herein, refersto amount, in picograms, of DNA contained within a haploid nucleus ofone half the amount in a diploid somatic cells of a eukaryotic organism.In some cases, the terms C-value and genome size are usedinterchangeably, however in polyploids the C-value can represent two ormore genomes contained within the same nucleus. In Oxytricha, which hasa unique genetic system, they play a critical role in development. Theyare also very useful to researchers as a means to alter DNA inside aliving organism. In some alternatives, a gene delivery polynucleotidefor stable insertion of a nucleic acid into a gene, wherein the nucleicacid for insertion is flanked by inverted terminal repeat gene sequencesin the gene delivery polynucleotide and wherein the gene deliverypolynucleotide is selectable, the gene delivery polynucleotide, isprovided. In some alternatives, the gene delivery polynucleotidecomprises a transposon.

The “Sleeping Beauty transposon system” as described herein, is composedof a Sleeping Beauty (SB) transposase and a transposon that was designedin 1997 to insert specific sequences of DNA into genomes of vertebrateanimals. DNA transposons can translocate from one DNA site to another ina simple, cut-and-paste manner. Transposition is a precise process inwhich a defined DNA segment is excised from one DNA molecule and movedto another site in the same or different DNA molecule or genome.

An SB transposase can insert a transposon into a TA dinucleotide basepair in a recipient DNA sequence. The insertion site can be elsewhere inthe same DNA molecule, or in another DNA molecule (or chromosome). Inmammalian genomes, including humans, there are approximately 200 millionTA sites. The TA insertion site is duplicated in the process oftransposon integration. This duplication of the TA sequence is ahallmark of transposition and used to ascertain the mechanism in someexperiments. The transposase can be encoded either within the transposonor the transposase can be supplied by another source, in which case thetransposon becomes a non-autonomous element.

In some alternatives, a gene delivery polynucleotide for stableinsertion of a nucleic acid into a gene, wherein the nucleic acid forinsertion is flanked by inverted terminal repeat gene sequences in thegene delivery polynucleotide and wherein the gene deliverypolynucleotide is selectable, the gene delivery polynucleotide, isprovided. In some alternatives, the gene delivery polynucleotidecomprises a transposon. In some alternatives, the transposon is aSleeping Beauty transposon. In some alternatives, the nucleic acid to beinserted is a Sleeping Beauty transposon flanked by inverted terminalrepeat gene sequences.

In some alternatives, the gene delivery polynucleotide for stableinsertion of nucleic acid is a minicircle. In some alternatives, thegene delivery polynucleotide for stable insertion of nucleic acidcomprises a Sleeping Beauty transposon. In some alternatives, methods ofgenerating engineered multiplexed T-cells are provided. In somealternatives, the method comprises delivering a Sleeping Beautytransposase to a cell. In some alternatives, methods of increasingprotein production in a T-cell are provided. In some alternatives, themethod comprises providing a vector encoding a Sleeping Beautytransposase. In some alternatives, the method comprises delivering avector encoding a Sleeping Beauty transposase to a cell.

“Codon optimization” as described herein, refers to the design processof altering codons to codons known to increase maximum proteinexpression efficiency in a desired cell. In some alternatives, codonoptimization is described, wherein codon optimization can be performedby using algorithms that are known to those skilled in the art to createsynthetic genetic transcripts optimized for high protein yield. Programscontaining alogorithms for codon optimization are known to those skilledin the art. Programs can include, for example, OptimumGene™, GeneGPS®algorithms, etc. Additionally synthetic codon optimized sequences can beobtained commercially for example from Integrated DNA Technologies andother commercially available DNA sequencing services. In somealternatives, a gene delivery polynucleotide for stable insertion of anucleic acid into a gene, wherein the nucleic acid for insertion isflanked by inverted terminal repeat gene sequences in the gene deliverypolynucleotide and wherein the gene delivery polynucleotide isselectable, is provided. In some alternatives, the gene deliverypolynucleotides are described, wherein the genes for the complete genetranscript are codon optimized for expression in humans. In somealternatives, the genes are optimized to have selected codonsspecifically for maximal protein expression in human cells, which canincrease the concentration of proteins or CARs of a T-cell.

Codon optimization can be performed to reduce the occurrence ofsecondary structure in a polynucleotide, as well. In some alternatives,codon optimization can also be performed to reduce the total GC/ATratio. Strict codon optimization can also lead to unwanted secondarystructure or an undesirable GC content that leads to secondarystructure. As such the secondary structures affect transcriptionalefficiency. Programs such as GeneOptimizer can be used after codon usageoptimization, for secondary structure avoidance and GC contentoptimization. These additional programs can be used for furtheroptimization and troubleshooting after an initial codon optimization tolimit secondary structures that may occur after the first round ofoptimization. Alternative programs for optimization are known to thoseskilled in the art. In some alternatives, a gene delivery polynucleotidefor stable insertion of a nucleic acid into a gene, wherein the nucleicacid for insertion is flanked by inverted terminal repeat gene sequencesin the gene delivery polynucleotide and wherein the gene deliverypolynucleotide is selectable, provided. In some alternatives, the genedelivery polynucleotide comprises sequences that are codon optimized forexpression in humans and/or to remove secondary structure and/or toreduce the total GC/AT ratio. In some alternatives, the sequences areoptimized for secondary structure avoidance. In some alternatives, thesequences are optimized to reduce the total GC/AT ratio.

In some alternatives, a method of generating engineered multiplexedT-cells for adoptive T-cell immunotherapy is provided. In the broadestsense, the method can comprise providing the gene deliverypolynucleotide of any one of the alternatives described herein,introducing the gene delivery polynucleotide into a T-cell, providing avector encoding a Sleeping Beauty transposase, introducing the vectorencoding the Sleeping Beauty transposase into the T-cell, selecting thecells comprising the gene delivery polynucleotide, wherein selectingcomprises a first round of selection and a second round of selection,wherein the first round of selection comprises adding a selectionreagent at a first concentration range and the second round of selectioncomprises adding the selection reagent at a second concentration range,wherein the second concentration range is higher than the firstconcentration range and, wherein the second concentration range is atleast 1.5 fold higher than that of the first concentration range, andisolating the T-cells expressing a phenotype under selective pressure.In some alternatives, the selection reagent is MTX.

Adoptive Immunotherapy for Cancer or a Viral Disease.

The premise of adoptive immunotherapy for cancer is transferring apatient's own tumor-specific T-cells into patients to facilitate thedestruction of malignant cells. T-cells can be genetically-engineered torecognize tumor-specific antigens and exert cytotoxic activity againstcancer cells. A method of adoptive immunotherapy for cancer is toisolate patient T-cells and introduce tumor recognition capability byexpressing chimeric antigen receptors (CARs), membrane proteins thatcontain an extracellular tumor-binding domain linked to an intracellularsignaling domain via a transmembrane segment. “Adoptive immunotherapy”or “T-cell adoptive transfer” refers to use of T-cell based cytotoxicresponse to attack cancer cells or specific cell targets. T-cells thathave a natural or genetically engineered reactivity to a patient'scancer can be generated in vitro and then transferred back into thesubject in need. Without being limiting, an example of adoptive transfercan be achieved by removing T-cells from a subject that has cancer or aviral disease and these T cells can be genetically engineered to expressreceptors specific for biomarkers found on a cancer cell or virus suchthat the genetically engineered T cells attack the cancer cells or virusor virus infected cells once the genetically engineered T-cells aretransferred back into the subject. In some alternatives, a method ofgenerating engineered multiplexed T-cells for adoptive T-cellimmunotherapy is provided. In some alternatives, methods of targetingmalignant cells for destruction are provided. In some alternatives amethod of treating, inhibiting, or ameliorating a cancer or a viraldisease in a subject is provided. In some alternatives the method oftreating, inhibiting, or ameliorating a cancer or a viral disease in asubject comprises administering to the subject an engineered multiplexedT-cells for adoptive T-cell immunotherapy. In some alternatives, thesubject is human.

The co-integration of additional genes can further increase theanti-tumor or antiviral activity of CAR-expressing T-cells.Comprehensive T-cell activation requires, in addition to initial tumoror viral recognition and signal initiation by CAR, engagement ofcostimulatory and cytokine receptors, which may not be present withinthe immunosuppressive environment of the tumor or the viral infectedsubject. To address this immunosuppressive environment of the tumor, forexample, expression of co-stimulatory ligands such as CD80 and 4-1BBL inengineered, CAR-expressing T-cells can result in greater T-cellexpansion due to auto-co-stimulation compared to expression ofco-stimulatory ligands on tumor cells. Another challenge in T-cellimmunotherapy is cell survival after infusion into patients. Inducedexpression of anti-apoptotic proteins has been shown to improve in vivosurvival of T-cells. Tumor homing and infiltration can be increased byintroduction of chemokine receptors in engineered T-cells and thisapproach can be especially useful for tumors that express chemokinesthat are not normally recognized by T-cells. Finally, T-cells can beengineered to better resist the immunosuppressive tumor microenvironmentor the immunocompromised virally infected subject through, for example,induced cytokine expression. Thus, methods to rapidly generateengineered T-cells expressing multiple transgenes are important andadvantageous for clinical translation of T-cell immunotherapy. In somealternatives, methods of generating engineered multiplexed T-cells foradoptive T-cell immunotherapy are provided. In some alternatives, theT-cells express chimeric antigen receptors. In some alternatives,T-cells expressing chimeric antigen receptors are engineered to expressco-stimulatory ligands. In some alternatives, the T-cells expressingchimeric antigen receptors express co-stimulatory ligands. In somealternatives the co-stimulatory ligands are CD80. In some alternatives,the co-stimulatory ligands are 4-1BBL.

Adoptive cell transfer can refer to the transfer of cells,immune-derived cells, back into the same patient or into a differentrecipient host. For isolation of immune cells for adoptive transfer,blood can be drawn into tubes containing anticoagulant and the PBM(buffy coat) cells are isolated, typically by density barriercentrifugation. In T-cell based therapies, the cells can be expanded invitro using cell culture methods relying heavily on the immunomodulatoryaction of interleukin-2 and returned to the patient in large numbersintravenously in an activated state. Anti-CD3 antibody can be used topromote the proliferation of T-cells in culture. Research intointerleukin-21 indicates that it can also play an important role inenhancing the efficacy of T-cell based therapies prepared in vitro.Cells used in adoptive cell transfer can be used to deliver geneticallymodified lymphocytes, using recombinant DNA technology to achieve anynumber of goals. In alternatives described herein, adoptive celltransfer is used to transfer cells into a subject, wherein the cells areCAR expressing lymphocytes. In some alternatives, CAR expressinglymphocytes are host cells in methods for generating engineeredmultiplexed T-cells for adoptive T-cell immunotherapy. In somealternatives, the method comprises providing the gene deliverypolynucleotide of the alternatives described herein, introducing thegene delivery polynucleotide into a T-cell, providing a vector encodinga Sleeping Beauty transposase, introducing the vector encoding theSleeping Beauty transposase into the T-cell, selecting the cellscomprising the gene delivery polynucleotide, wherein selecting comprisesa first round of selection and a second round of selection, wherein thefirst round of selection comprises adding a selection reagent at a firstconcentration range and the second round of selection comprises addingthe selection reagent at a second concentration range, wherein thesecond concentration range is higher than the first concentration rangeand, wherein the second concentration range is at least 1.5 fold higherthan that of the first concentration range, and isolating the T-cellsexpressing a phenotype under selective pressure. In some alternatives,the gene delivery polynucleotide comprises a sequence for aco-stimulatory ligand. In some alternatives, the gene deliverypolynucleotide comprises a sequence for a chimeric antigen receptor. Insome alternatives, the T-cell expresses a CAR. In alternatives describedherein, the CAR expressing lymphocytes are genetically modified byminicircles wherein the minicircles comprise Sleeping Beautytransposons. In some alternatives, the selection reagent is MTX.

By way of example and not of limitation, genetically engineered T-cellscan be created by infecting patient's cells with a transferring virusthat contain a copy of a T-cell receptor (TCR) gene that is specializedto recognize, for example, tumor or viral antigens. It is important thatthe transferring virus is not able to reproduce within the cell however,but should integrate into the human genome. This is beneficial as newTCR gene remains stable in the T-cell. A patient's own T-cells areexposed to these transferring viruses and then are expandednon-specifically or stimulated using the genetically engineered TCR. Thecells are then transferred back into the patient and are ready to mountan immune response against the tumor, virus, or viral infected cell. Theuse of adoptive cell transfer with genetically engineered T-cells is apromising new approach for the treatment of a variety of cancers orviral infections. In some alternatives, methods of adoptiveimmunotherapy for cancer are provided. In some alternatives, methods ofadoptive immunotherapy for viral infections are provided.

The method of making genetically engineered T-cells by using a viralvector can have several drawbacks. Genetic modification of T-cells istypically accomplished using γ-retroviral or lentiviral vectors. Whileeffective, drawbacks include cost of production, limited gene packagingcapacity, and potential safety issues. Plasmids containing transposonsystems such as Sleeping Beauty (SB) or piggyBac offer a non-viralapproach for stably introducing genes into T-cells. Recently, thepiggyBac system was used to produce stably-transfected mammalian cellsexpressing multiple transgenes of interest by delivery of multipletransposons. The SB system, first reactivated for mammalian cell use byIvics and coworkers, has been used as the gene delivery modality inclinical trials of T-cell immunotherapy. Gene integration by SB hasweaker preference for transcriptional units and their regulatorysequences compared to the γ-retroviral and lentiviral vectors and istherefore considered to be safer. In some alternatives described herein,genetic modification by minicircles comprising the Sleeping Beautysystem are contemplated. In some alternatives described herein, geneticmodification by minicircles comprising the piggyBac system arecontemplated. In some alternatives described herein, geneticmodification by minicircles comprising the Sleeping Beauty system arecontemplated.

Minicircles are particularly attractive as transfection platforms forthree reasons. First, the transfection efficiency of minicircles byelectroporation is superior to that of their plasmid analogues. Second,transposition efficiency is higher in minicircles due to the shorterdistance between the two transposon ends, which has been shown to affecttransposase efficiency. Finally, as cell viability after nucleofectiondecreases with increasing construct size, minicircles are moreadvantageous given their smaller size compared to their analogousplasmids. To further improve transposition efficiency, the optimizedSB100X hyperactive transposase developed by Izsvak et al. (Nature Genet.2009, 41, 753-761; incorporated by reference in its entirety) can beused in combination with the T3 generation of SB previously by Yant etal (Mol. Cell. Biol. 2004, 24, 9239-9247; incorporated by reference inits entirety). In several alternatives described herein, methods formaking a genetically modified T-cell for adoptive cell transfer arecontemplated. In some alternatives, the methods comprise introducing aminicircle into a T-cell. In some alternatives, the introductioncomprises electroporation delivery.

Another challenge in T-cell immunotherapy is cell survival afterinfusion into patients. Induced expression of anti-apoptotic proteinshas been shown to improve in vivo survival of T-cells. Tumor homing andinfiltration has been increased by introduction of chemokine receptorsin engineered T-cells; this approach can be especially useful for tumorsthat express chemokines that are not normally recognized by T-cells.Finally, T-cells can be engineered to better resist theimmunosuppressive tumor microenvironment through, for example, inducedcytokine expression. Thus, methods to rapidly generate engineeredT-cells expressing multiple transgenes are important and advantageousfor clinical translation of T-cell immunotherapy. In some alternativesdescribed herein, methods of introducing co-integration of additionalgenes for co-integration to further increase the anti-tumor activity ofCAR-expressing T-cells are contemplated. In some alternatives, theadditional genes encode co-stimulatory ligands. In some alternatives,the co-stimulatory ligand is CD80. In some alternatives, theco-stimulatory ligand is 4-1BBL. In some alternatives, the additionalgenes encode anti-apoptotic proteins. In some alternatives theadditional genes encode chemokine receptors.

In some alternatives, methods of generating engineered multiplexedT-cells for adoptive T-cell immunotherapy are provided. In the broadestsense, the method can comprise providing the gene deliverypolynucleotide of any of the alternatives described herein, introducingthe gene delivery polynucleotide into a T-cell, providing a vectorencoding a Sleeping Beauty transposase, introducing the vector encodingthe Sleeping Beauty transposase into the T-cell, selecting the cellscomprising the gene delivery polynucleotide, wherein selecting comprisesa first round of selection and a second round of selection, wherein thefirst round of selection comprises adding a selection reagent at a firstconcentration range and the second round of selection comprises addingthe selection reagent at a second concentration range, wherein thesecond concentration range is higher than the first concentration rangeand, wherein the second concentration range is at least 1.5 fold higherthan that of the first concentration range, and isolating the T-cellsexpressing a phenotype under selective pressure. In some alternatives,the T-cells are chimeric antigen receptor (CAR) expressing T-cells. Insome alternatives, the selection reagent is MTX.

In some alternatives, methods of increasing protein production in aT-cell are provided. In the broadest sense, the method can compriseproviding the gene delivery polynucleotide of any of the alternativesdescribed herein, introducing the gene delivery polynucleotide into aT-cell, providing a vector encoding a Sleeping Beauty transposase,introducing the vector encoding the Sleeping Beauty transposase into theT-cell, selecting the cells comprising the gene delivery polynucleotide,wherein selecting comprises a first round of selection and a secondround of selection, wherein the first round of selection comprisesadding a selection reagent at a first concentration range and the secondround of selection comprises adding the selection reagent at a secondconcentration range, wherein the second concentration range is higherthan the first concentration range and, wherein the second concentrationrange is at least 1.5 fold higher than that of the first concentrationrange, and isolating the T-cells expressing a phenotype under selectivepressure. In some alternatives, the selection reagent is MTX. In somealternatives, the T-cells are chimeric antigen receptor (CAR) expressingT-cells.

As described herein, an alternative of the system comprises anengineered, non-viral gene delivery system comprising three keyfeatures: (1) Sleeping Beauty transposon system for stable geneexpression, (2) minicircles for enhanced transfection, and (3) a doublemutant of human dihydrofolate reductase (DHFRdm) as a selectionmechanism (FIG. 1).

Minicircles are particularly attractive as transfection platforms forthree reasons. First, the transfection efficiency of minicircles byelectroporation is superior to that of their plasmid analogues. Second,transposition efficiency is higher in minicircles due to the shorterdistance between the two transposon ends, which has been shown to affecttransposase efficiency. Finally, as cell viability after nucleofectiondecreases with increasing construct size, minicircles are more desirablegiven their smaller size compared to their analogous plasmids. Tofurther improve transposition efficiency, the optimized SB100Xhyperactive transposase developed by Izsvak et al. (Nature Genet. 2009,41, 753-761; incorporated herein by reference in its entirety) was usedin combination with the T3 generation of SB transposon previously byYant et al (Mol. Cell. Biol. 2004, 24, 9239-9247; incorporated herein byreference in its entirety). In some alternatives described herein,genetic modification of T-cells is performed using minicircles. In somealternatives, the minicircles comprise transposons. In somealternatives, the transposons comprise Sleeping Beauty transposons. Insome alternatives, an optimized SB100X hyperactive transposase is usedin combination with a T3 generation of SB transposon.

A selection mechanism for rapid selection of engineered T-cells can alsobe employed. The double mutant of human dihydrofolate reductase (DHFRdm,with amino acid mutations L22F and F31S) exhibits a 15,000-fold reducedaffinity for methotrexate, a potent inhibitor of DHFR that results inblockade of thymidylate and purine synthesis. Expression of DHFRdm inT-cells imparts MTX resistance without compromising proliferativeability, expression of T-cell markers, or cytolytic ability. Additionaladvantages of this selection system include availability of clinicalgrade MTX, the use of a non-genotoxic drug, and the small gene size ofDHFRdm (561 bp). Therefore, MTX can be used as a selection mechanism toselectively amplify SB-transduced cells. In some alternatives, theminicircles comprise a genetic sequence encoding a double mutant ofhuman dihydrofolate reductase. In some alternatives, a selection methodfor rapid selection of engineered T-cells is provided. In somealternatives, the selection method comprises contacting engineeredT-cells with clinical grade methotrexate. In some alternatives, theT-cells comprise a minicircle wherein the minicircle comprises asequence for a double mutant of human dihydrofolate reductase. In somealternatives, the double mutant of human dihydrofolate reductaseexhibits a 15,000 fold or about 15,000 fold reduced specificity formethotrexate. In some alternatives, methotrexate can be used to contactthe T-cells for selectively amplifying cells transduced withminicircles, wherein the minicircles comprise a sequence for the doublemutant of human dihydrofolate reductase. In some alternatives, the geneencoding the double mutant of human dihydrofolate reductase comprisesthe DNA sequence:ATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACTTCCCCTGGCCACCGCTCAGGAATGAATCCAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAAGGAGCTCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAGAATGATTAA (SEQ ID NO: 2). In some alternatives, the doublemutant of human dihydrofolate reductase comprises the protein sequence:MVGSLNCIVA VSQNMGIGKN GDFPWPPLRN ESRYFQRMTT TSSVEGKQNL VIMGKKTWFSIPEKNRPLKG RINLVLSREL KEPPQGAHFL SRSLDDALKL TEQPELANKV DMVWIVGGSSVYKEAMNHPG HLKLFVTRIM QDFESDTFFP EIDLEKYKLL PEYPGVLSDV QEEKGIKYKFEVYEKND (SEQ ID NO: 3).

Stable transfer of up to three transgenes into the H9 T-cell line usingmultiplexed delivery of minicircles containing SB transposons followedby methotrexate (MTX) selection can be performed. Cells with highernumber of gene integrations can be preferentially obtained by increasingselection pressure with MTX. Using a two-step selection method throughtwo successive MTX selection rounds, 50% of cells expressing threetransgene products can be obtained. In some alternatives, a method ofstably transferring transgenes into a cell line is provided. In somealternatives, a method of introducing minicircles into a cell line isprovided. In some alternatives, the minicircles comprise Sleeping Beautytransposons. In some alternatives, the method further comprisesincreasing selection pressure with methotrexate, wherein increasing theselection pressure comprises contacting the cell line with increasingconcentrations of methotrexate. In some alternatives, the two rounds ofmethotrexate selection are performed.

Additional Alternatives

In some alternatives, a gene delivery polynucleotide for stableinsertion of a nucleic acid into a gene, wherein the nucleic acid forinsertion is flanked by inverted terminal repeat gene sequences in thegene delivery polynucleotide and wherein the gene deliverypolynucleotide is selectable, the gene delivery polynucleotide, isprovided. In the broadest sense, the gene delivery polynucleotidecomprises a first sequence, wherein the first sequence comprises a firstinverted terminal repeat gene sequence, a second sequence, wherein thesecond sequence comprises a second inverted terminal repeat genesequence, a third sequence, wherein the third sequence comprises apromoter region sequence, a fourth sequence, wherein the fourth sequencecomprises at least one gene encoding a protein, and wherein the fourthsequence is optimized, a fifth sequence, wherein the fifth sequencecomprises at least one selectable marker cassette encoding a doublemutant of dihydrofolate reductase, wherein the double mutant ofdihydrofolate reductase has a 15,000 fold or about 15,000 fold reducedaffinity for methotrexate, wherein the methotrexate can be used as aselection mechanism to selectively amplify cells transduced with thegene delivery polynucleotide and wherein the fifth sequence isoptimized, a sixth sequence, wherein the sixth sequence comprises afirst attachment site (attP), and a seventh sequence, wherein theseventh sequence comprises a second attachment site (attB); wherein eachof the first sequence, second sequence, third sequence, fourth sequence,fifth sequence, sixth sequence, and seventh sequence have a 5′ terminusand a 3′ terminus, and wherein the 3′ terminus of the first sequencecomprising the first inverted terminal repeat gene sequence is adjacentto the 5′ terminus of the third sequence, the 3′ terminus of the thirdsequence is adjacent to the 5′ terminus of the fourth sequence, the 3′terminus of the fourth sequence is adjacent to the 5′ terminus of thefifth sequence and the 3′ terminus of the fifth sequence is adjacent tothe 5′ terminus of the second sequence comprising a second invertedterminal repeat. In some alternatives, the gene encoding the doublemutant of human dihydrofolate reductase comprises the DNA sequence:ATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACTTCCCCTGGCCACCGCTCAGGAATGAATCCAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAAGGAGCTCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAGAATGATTAA (SEQ ID NO: 2). In some alternatives, the doublemutant of human dihydrofolate reductase comprises the protein sequence:MVGSLNCIVA VSQNMGIGKN GDFPWPPLRN ESRYFQRMTT TSSVEGKQNL VIMGKKTWFSIPEKNRPLKG RINLVLSREL KEPPQGAHFL SRSLDDALKL TEQPELANKV DMVWIVGGSSVYKEAMNHPG HLKLFVTRIM QDFESDTFFP EIDLEKYKLL PEYPGVLSDV QEEKGIKYKFEVYEKND (SEQ ID NO: 3). In some alternatives, the gene deliverypolynucleotide is circular. In some alternatives, the gene deliverypolynucleotide is at least 1 kB to 6 kB. In some alternatives, the genedelivery polynucleotide is a minicircle. In some alternatives, thepromoter region comprises an EF1 promoter sequence. In somealternatives, the fourth sequence comprises one, two, three, four, orfive genes that encode proteins. In some alternatives, the fourthsequence is codon optimized to reduce the total GC/AT ratio of thefourth sequence. In some alternatives, the fourth sequence is aconsensus sequence generated from a plurality of nucleic acids thatencode a plurality of related proteins, such as a plurality of antibodybinding domains, which are specific for the same epitope. In somealternatives, the fifth sequence is codon optimized to reduce the totalGC/AT ratio of the fourth sequence. In some alternatives, the codonoptimization and/or consensus sequence is generated by comparing thevariability of sequence and/or nucleobases utilized in a plurality ofrelated sequences. In some alternatives, the protein is a protein fortherapy. In some alternatives, the protein comprises an antibody or aportion thereof. In some alternatives, the double mutant ofdihydrofolate reductase comprises amino acid mutations of L22F and F31S.In some alternatives, the minicircle comprises a sequence for the doublemutant of dihydrofolate reductase, the sequence comprising the DNAsequence ATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACTTCCCCTGGCCACCGCTCAGGAATGAATCCAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAAGGAGCTCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAGAATGATTAA (SEQ ID NO: 2). In some alternatives, the doublemutant of dihydrofolate reductase comprises the protein sequence:MVGSLNCIVA VSQNMGIGKN GDFPWPPLRN ESRYFQRMTT TSSVEGKQNL VIMGKKTWFSIPEKNRPLKG RINLVLSREL KEPPQGAHFL SRSLDDALKL TEQPELANKV DMVWIVGGSSVYKEAMNHPG HLKLFVTRIM QDFESDTFFP EIDLEKYKLL PEYPGVLSDV QEEKGIKYKFEVYEKND (SEQ ID NO: 3).

In some alternatives, a method of generating engineered multiplexedT-cells for adoptive T-cell immunotherapy is provided. In the broadestsense, the method can comprise providing the gene deliverypolynucleotide of any of the alternatives described herein, introducingthe gene delivery polynucleotide into a T-cell, providing a vectorencoding a Sleeping Beauty transposase, introducing the vector encodingthe Sleeping Beauty transposase into the T-cell, selecting the cellscomprising the gene delivery polynucleotide, wherein selecting comprisesa first round of selection and a second round of selection, wherein thefirst round of selection comprises adding a selection reagent at a firstconcentration range and the second round of selection comprises addingthe selection reagent at a second concentration range, wherein thesecond concentration range is higher than the first concentration rangeand, wherein the second concentration range is at least 1.5 fold higherthan that of the first concentration range, and isolating the T-cellsexpressing a phenotype under selective pressure. In some alternatives,introducing is performed by electroporation. In some alternatives, theselecting is performed by increasing selective pressure through theselective marker cassette. In some alternatives, the selection reagentcomprises an agent for selection. In some alternatives, the agent forselection is methotrexate. In some alternatives, the first concentrationrange is at least 50 nM-100 nM and the second concentration range is atleast 75 to 150 nM. In some alternatives, the first concentration is 50nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100 nM or any concentration that isbetween a range of concentrations defined by any two of theaforementioned concentrations, and the second concentration range is 75nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, or 150 nM orany concentration that is between a range of concentrations defined byany two of the aforementioned concentrations. In some alternatives, thefirst concentration range is at least 75 nM-150 nM and the secondconcentration range is at least 112.5 nM to 225 nM. In somealternatives, the first concentration is 75 nM, 85 nM, 95 nM, 105 nM,115 nM, 125 nM, 135 nM, 145 nM, or 150 nM or any concentration that isbetween a range of concentrations defined by any two of theaforementioned concentrations, and the second concentration range is 112nM, 122 nM, 132 nM, 142 nM, 152 nM, 162 nM, 172 nM, 182 nM, 192 nM, 202nM, 212 nM, or 225 nM or any concentration that is between a range ofconcentrations defined by any two of the aforementioned concentrations.In some alternatives, the first concentration range is at least 300nM-675 nM and the first concentration range is at least 450 nM to 1012nM. In some alternatives, the first concentration is 300 nM, 350 nM, 400nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, or 675 nM or anyconcentration that is between a range of concentrations defined by anytwo of the aforementioned concentrations, and the second concentrationrange is 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM,850 nM, 900 nM, 1000 nM, or 1012 nM or any concentration that is betweena range of concentrations defined by any two of the aforementionedconcentrations. In some alternatives, the first round of selectioncomprises exposing the T-cells to the selection agent for 2, 3, 4, 5, 6or 7 days before the second round of selection. In some alternatives,the second round of selection comprises exposing the T-cells to theselection agent for at least 2, 3, 4, 5, 6 or 7 days before isolation.

In some alternatives, a method of increasing protein production in acell is provided. In the broadest sense, the method can compriseproviding the gene delivery polynucleotide of any one of thealternatives described herein, introducing the gene deliverypolynucleotide into a T-cell, providing a vector encoding a SleepingBeauty transposase, introducing the vector encoding the Sleeping Beautytransposase into the T-cell, selecting the cells comprising the genedelivery polynucleotide, wherein selecting comprises a first round ofselection and a second round of selection, wherein the first round ofselection comprises adding a selection reagent at a first concentrationrange and the second round of selection comprises adding the selectionreagent at a second concentration range, wherein the secondconcentration range is higher than the first concentration range and,wherein the second concentration range is at least 1.5 fold higher thanthat of the first concentration range, and isolating the T-cellsexpressing a phenotype under selective pressure. In some alternatives,introducing is performed by electroporation. In some alternatives,selecting is performed by increasing selective pressure through theselective marker cassette. In some alternatives, the selection reagentcomprises an agent for selection. In some alternatives, the agent forselection is methotrexate. In some alternatives, the low or firstconcentration range is at least 50 nM-100 nM and the higher or secondconcentration range is at least 75 to 150 nM. In some alternatives, thelow or first concentration range is at least 75 nM-150 nM and the higheror second concentration range is at least 112.5 nM to 225 nM. In somealternatives, the low or first concentration range is at least 300nM-675 nM and the higher or second concentration range is at least 450nM to 1012 nM. In some alternatives, the first round of selectioncomprises exposing the T-cells to the selection agent for 2, 3, 4, 5, 6or 7 days before the second round of selection. In some alternatives,the second round of selection comprises exposing the T-cells to theselection agent for at least 2, 3, 4, 5, 6 or 7 days before isolation.

In some alternatives, an engineered multiplexed T-cell for adoptiveT-cell immunotherapy generated by any one of the methods of is provided.In some alternatives, the engineered multiplexed T-cells for adoptiveT-cell immunotherapy is generated by a method, wherein the methodcomprises providing a gene delivery polynucleotide, introducing the genedelivery polynucleotide into a T-cell, providing a vector encoding aSleeping Beauty transposase, introducing the vector encoding theSleeping Beauty transposase into the T-cell, selecting the cellscomprising the gene delivery polynucleotide wherein selecting comprisesa first round of selection and a second round of selection, wherein thefirst round of selection comprises adding a selection reagent at a firstconcentration range and the second round of selection comprises addingthe selection reagent at a second concentration range, wherein thesecond concentration range is higher than the first concentration rangeand, wherein the second concentration range is at least 1.5 fold higherthan that of the first concentration range and isolating the T-cellsexpressing a phenotype under selective pressure. In some alternatives,the gene delivery polynucleotide comprises a first sequence, wherein thefirst sequence comprises a first inverted terminal repeat gene sequence,a second sequence, wherein the second sequence comprises a secondinverted terminal repeat gene sequence, a third sequence, wherein thethird sequence comprises a promoter region sequence, a fourth sequence,wherein the fourth sequence comprises at least one gene encoding aprotein, and wherein the fourth sequence is optimized, a fifth sequence,wherein the fifth sequence comprises at least one selectable markercassette encoding a double mutant of dihydrofolate reductase, whereinthe double mutant of dihydrofolate reductase has a 15,000 fold or about15,000 fold reduced affinity for methotrexate, wherein the methotrexatecan be used as a selection mechanism to selectively amplify cellstransduced with the gene delivery polynucleotide and wherein the fifthsequence is optimized, a sixth sequence, wherein the sixth sequencecomprises a first attachment site (attP) and a seventh sequence, whereinthe seventh sequence comprises a second attachment site (attB) whereineach of the first sequence, second sequence, third sequence, fourthsequence, fifth sequence, sixth sequence, and seventh sequence have a 5′terminus and a 3′ terminus, and wherein the 3′ terminus of the firstsequence comprising the first inverted terminal repeat gene sequence isadjacent to the 5′ terminus of the third sequence, the 3′ terminus ofthe third sequence is adjacent to the 5′ terminus of the fourthsequence, the 3′ terminus of the fourth sequence is adjacent to the 5′terminus of the fifth sequence and the 3′ terminus of the fifth sequenceis adjacent to the 5′ terminus of the second sequence comprising asecond inverted terminal repeat. In some alternatives, the gene encodingthe double mutant of human dihydrofolate reductase comprises the DNAsequence: ATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACTTCCCCTGGCCACCGCTCAGGAATGAATCCAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAAGGAGCTCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAGAATGATTAA (SEQ ID NO: 2). In some alternatives, the doublemutant of human dihydrofolate reductase comprises the protein sequence:MVGSLNCIVA VSQNMGIGKN GDFPWPPLRN ESRYFQRMTT TSSVEGKQNL VIMGKKTWFSIPEKNRPLKG RINLVLSREL KEPPQGAHFL SRSLDDALKL TEQPELANKV DMVWIVGGSSVYKEAMNHPG HLKLFVTRIM QDFESDTFFP EIDLEKYKLL PEYPGVLSDV QEEKGIKYKFEVYEKND (SEQ ID NO: 3). In some alternatives, the gene deliverypolynucleotide is circular. In some alternatives, the gene deliverypolynucleotide is at least 1 kB to 5 kB. In some alternatives, the genedelivery polynucleotide is a minicircle. In some alternatives, thepromoter region comprises an EF1 promoter sequence. In somealternatives, the fourth sequence comprises one, two, three, four, orfive genes that encode proteins. In some alternatives, the fourthsequence is codon optimized to reduce the total GC/AT ratio of thefourth sequence. In some alternatives, the fourth sequence is optimizedby codon optimization for expression in humans. In some alternatives,the fourth sequence is a consensus sequence generated from a pluralityof nucleic acids that encode a plurality of related proteins. In somealternatives, the fourth sequence is a consensus sequence generated froma plurality of nucleic acids that encode a plurality of relatedproteins, such as a plurality of antibody binding domains, which arespecific for the same epitope. In some alternatives, the plurality ofrelated proteins comprise a plurality of antibody binding domains,wherein the plurality of antibody binding domains are specific for thesame epitope. In some alternatives, the fifth sequence is codonoptimized to reduce the total GC/AT ratio of the fifth sequence. In somealternatives, the fifth sequence is optimized by codon optimization forexpression in humans. In some alternatives, the protein is a protein fortherapy. In some alternatives, the codon optimization and/or consensussequence is generated by comparing the variability of sequence and/ornucleobases utilized in a plurality of related sequences. In somealternatives, the protein comprises an antibody or a portion thereof,which may be humanized. In some alternatives, the double mutant ofdihydrofolate reductase comprises amino acid mutations of L22F and F31S.In some alternatives, the double mutant of dihydrofolate reductasecomprises amino acid mutations of L22F and F31S. In some alternatives,the introducing is performed by electroporation. In some alternatives,the selecting is performed by increasing selective pressure through theselective marker cassette. In some alternatives, the selection reagentcomprises an agent for selection. In some alternatives, the agent forselection is methotrexate. In some alternatives, the first concentrationrange is at least 50 nM-100 nM and the second concentration range is atleast 75 to 150 nM. In some alternatives, the first concentration is 50nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100 nM or any concentration that isbetween a range of concentrations defined by any two of theaforementioned concentrations, and the second concentration range is 75nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, or 150 nM orany concentration that is between a range of concentrations defined byany two of the aforementioned concentrations. In some alternatives, thefirst concentration range is at least 75 nM-150 nM and the secondconcentration range is at least 112.5 nM to 225 nM. In somealternatives, the first concentration is 75 nM, 85 nM, 95 nM, 105 nM,115 nM, 125 nM, 135 nM, 145 nM, or 150 nM or any concentration that isbetween a range of concentrations defined by any two of theaforementioned concentrations, and the second concentration range is 112nM, 122 nM, 132 nM, 142 nM, 152 nM, 162 nM, 172 nM, 182 nM, 192 nM, 202nM, 212 nM, or 225 nM or any concentration that is between a range ofconcentrations defined by any two of the aforementioned concentrations.In some alternatives, the first concentration range is at least 300nM-675 nM and the first concentration range is at least 450 nM to 1012nM. In some alternatives, the first concentration is 300 nM, 350 nM, 400nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, or 675 nM or anyconcentration that is between a range of concentrations defined by anytwo of the aforementioned concentrations, and the second concentrationrange is 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM,850 nM, 900 nM, 1000 nM, or 1012 nM or any concentration that is betweena range of concentrations defined by any two of the aforementionedconcentrations. In some alternatives, the first round of selectioncomprises exposing the T-cells to the selection agent for 2, 3, 4, 5, 6or 7 days before the second round of selection. In some alternatives,the second round of selection comprises exposing the T-cells to theselection agent for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or14 days or any time that is between a range of times defined by any twoof the aforementioned time points before isolation. In somealternatives, the gene delivery polynucleotide comprises a firstsequence, wherein the first sequence comprises a first inverted terminalrepeat gene sequence, a second sequence, wherein the second sequencecomprises a second inverted terminal repeat gene sequence, a thirdsequence, wherein the third sequence comprises a promoter regionsequence, a fourth sequence, wherein the fourth sequence comprises atleast one gene encoding a protein, and wherein the fourth sequence isoptimized, a fifth sequence, wherein the fifth sequence comprises atleast one selectable marker cassette encoding a double mutant ofdihydrofolate reductase, wherein the double mutant of dihydrofolatereductase has a 15,000 fold or about 15,000 fold reduced affinity formethotrexate, wherein the methotrexate can be used as a selectionmechanism to selectively amplify cells transduced with the gene deliverypolynucleotide and wherein the fifth sequence is optimized, a sixthsequence, wherein the sixth sequence comprises a first attachment site(attP) and a seventh sequence, wherein the seventh sequence comprises asecond attachment site (attB) wherein each of the first sequence, secondsequence, third sequence, fourth sequence, fifth sequence, sixthsequence, and seventh sequence have a 5′ terminus and a 3′ terminus, andwherein the 3′ terminus of the first sequence comprising the firstinverted terminal repeat gene sequence is adjacent to the 5′ terminus ofthe third sequence, the 3′ terminus of the third sequence is adjacent tothe 5′ terminus of the fourth sequence, the 3′ terminus of the fourthsequence is adjacent to the 5′ terminus of the fifth sequence and the 3′terminus of the fifth sequence is adjacent to the 5′ terminus of thesecond sequence comprising a second inverted terminal repeat. In somealternatives, the gene encoding the double mutant of human dihydrofolatereductase comprises the DNA sequence:ATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACTTCCCCTGGCCACCGCTCAGGAATGAATCCAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAAGGAGCTCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAGAATGATTAA (SEQ ID NO: 2). In some alternatives, the doublemutant of human dihydrofolate reductase comprises the protein sequence:MVGSLNCIVA VSQNMGIGKN GDFPWPPLRN ESRYFQRMTT TSSVEGKQNL VIMGKKTWFSIPEKNRPLKG RINLVLSREL KEPPQGAHFL SRSLDDALKL TEQPELANKV DMVWIVGGSSVYKEAMNHPG HLKLFVTRIM QDFESDTFFP EIDLEKYKLL PEYPGVLSDV QEEKGIKYKFEVYEKND (SEQ ID NO: 3). In some alternatives, the gene deliverypolynucleotide is circular. In some alternatives, the gene deliverypolynucleotide is at least 1 kB to 5 kB. In some alternatives, the genedelivery polynucleotide is a minicircle. In some alternatives, thepromoter region comprises an EF1 promoter sequence. In somealternatives, the fourth sequence comprises one, two, three, four, orfive genes that encode proteins. In some alternatives, the fourthsequence is codon optimized to reduce the total GC/AT ratio of thefourth sequence. In some alternatives, the fourth sequence is optimizedby codon optimization for expression in humans. In some alternatives,the fourth sequence is a consensus sequence generated from a pluralityof nucleic acids that encode a plurality of related proteins. In somealternatives, the fourth sequence is a consensus sequence generated froma plurality of nucleic acids that encode a plurality of relatedproteins, such as a plurality of antibody binding domains, which arespecific for the same epitope. In some alternatives, the plurality ofrelated proteins comprise a plurality of antibody binding domains,wherein the plurality of antibody binding domains are specific for thesame epitope. In some alternatives, the fifth sequence is codonoptimized to reduce the total GC/AT ratio of the fifth sequence. In somealternatives, the fifth sequence is optimized by codon optimization forexpression in humans. In some alternatives, the protein is a protein fortherapy. In some alternatives, the codon optimization and/or consensussequence is generated by comparing the variability of sequence and/ornucleobases utilized in a plurality of related sequences. In somealternatives, the protein comprises an antibody or a portion thereof,which may be humanized. In some alternatives, the double mutant ofdihydrofolate reductase comprises amino acid mutations of L22F and F31S.In some alternatives, the introducing is performed by electroporation.In some alternatives, the selecting is performed by increasing selectivepressure through the selective marker cassette. In some alternatives,the selection reagent comprises an agent for selection. In somealternatives, the agent for selection is methotrexate. In somealternatives, the first concentration range is at least 50 nM-100 nM andthe second concentration range is at least 75 to 150 nM. In somealternatives, the first concentration is 50 nM, 60 nM, 70 nM, 80 nM, 90nM, or 100 nM or any concentration that is between a range ofconcentrations defined by any two of the aforementioned concentrations,and the second concentration range is 75 nM, 80 nM, 90 nM, 100 nM, 110nM, 120 nM, 130 nM, 140 nM, or 150 nM or any concentration that isbetween a range of concentrations defined by any two of theaforementioned concentrations. In some alternatives, the firstconcentration range is at least 75 nM-150 nM and the secondconcentration range is at least 112.5 nM to 225 nM. In somealternatives, the first concentration is 75 nM, 85 nM, 95 nM, 105 nM,115 nM, 125 nM, 135 nM, 145 nM, or 150 nM or any concentration that isbetween a range of concentrations defined by any two of theaforementioned concentrations, and the second concentration range is 112nM, 122 nM, 132 nM, 142 nM, 152 nM, 162 nM, 172 nM, 182 nM, 192 nM, 202nM, 212 nM, or 225 nM or any concentration that is between a range ofconcentrations defined by any two of the aforementioned concentrations.In some alternatives, the first concentration range is at least 300nM-675 nM and the first concentration range is at least 450 nM to 1012nM. In some alternatives, the first concentration is 300 nM, 350 nM, 400nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, or 675 nM or anyconcentration that is between a range of concentrations defined by anytwo of the aforementioned concentrations, and the second concentrationrange is 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM,850 nM, 900 nM, 1000 nM, or 1012 nM or any concentration that is betweena range of concentrations defined by any two of the aforementionedconcentrations. In some alternatives, the first round of selectioncomprises exposing the T-cells to the selection agent for 2, 3, 4, 5, 6or 7 days before the second round of selection. In some alternatives,the second round of selection comprises exposing the T-cells to theselection agent for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or14 days or any time that is between a range of times defined by any twoof the aforementioned time points before isolation.

In some alternatives, a method of treating, inhibiting, or amelioratingcancer or a disease in a subject is provided, wherein the methodcomprises administering to the subject the modified or engineeredmultiplexed T-cell as described below. In some alternatives, theengineered multiplexed T-cells for adoptive T-cell immunotherapy isgenerated by a method, wherein the method comprises providing a genedelivery polynucleotide, introducing the gene delivery polynucleotideinto a T-cell, providing a vector encoding a Sleeping Beautytransposase, introducing the vector encoding the Sleeping Beautytransposase into the T-cell, selecting the cells comprising the genedelivery polynucleotide wherein selecting comprises a first round ofselection and a second round of selection, wherein the first round ofselection comprises adding a selection reagent at a first concentrationrange and the second round of selection comprises adding the selectionreagent at a second concentration range, wherein the secondconcentration range is higher than the first concentration range and,wherein the second concentration range is at least 1.5 fold higher thanthat of the first concentration range and isolating the T-cellsexpressing a phenotype under selective pressure. In some alternatives,the gene delivery polynucleotide comprises a first sequence, wherein thefirst sequence comprises a first inverted terminal repeat gene sequence,a second sequence, wherein the second sequence comprises a secondinverted terminal repeat gene sequence, a third sequence, wherein thethird sequence comprises a promoter region sequence, a fourth sequence,wherein the fourth sequence comprises at least one gene encoding aprotein, and wherein the fourth sequence is codon optimized forexpression in humans, a fifth sequence, wherein the fifth sequencecomprises at least one selectable marker cassette encoding a doublemutant of dihydrofolate reductase, wherein the double mutant ofdihydrofolate reductase has a 15,000 fold or about 15,000 fold reducedaffinity for methotrexate, wherein the methotrexate can be used as aselection mechanism to selectively amplify cells transduced with thegene delivery polynucleotide and wherein the fifth sequence is codonoptimized for expression in humans, a sixth sequence, wherein the sixthsequence comprises a first attachment site (attP) and a seventhsequence, wherein the seventh sequence comprises a second attachmentsite (attB) wherein each of the first sequence, second sequence, thirdsequence, fourth sequence, fifth sequence, sixth sequence, and seventhsequence have a 5′ terminus and a 3′ terminus, and wherein the 3′terminus of the first sequence comprising the first inverted terminalrepeat gene sequence is adjacent to the 5′ terminus of the thirdsequence, the 3′ terminus of the third sequence is adjacent to the 5′terminus of the fourth sequence, the 3′ terminus of the fourth sequenceis adjacent to the 5′ terminus of the fifth sequence and the 3′ terminusof the fifth sequence is adjacent to the 5′ terminus of the secondsequence comprising a second inverted terminal repeat. In somealternatives, the gene encoding the double mutant of human dihydrofolatereductase comprises the DNA sequence:ATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACTTCCCCTGGCCACCGCTCAGGAATGAATCCAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAAGGAGCTCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAGAATGATTAA (SEQ ID NO: 2). In some alternatives, the doublemutant of human dihydrofolate reductase comprises the protein sequence:MVGSLNCIVA VSQNMGIGKN GDFPWPPLRN ESRYFQRMTT TSSVEGKQNL VIMGKKTWFSIPEKNRPLKG RINLVLSREL KEPPQGAHFL SRSLDDALKL TEQPELANKV DMVWIVGGSSVYKEAMNHPG HLKLFVTRIM QDFESDTFFP EIDLEKYKLL PEYPGVLSDV QEEKGIKYKFEVYEKND (SEQ ID NO: 3). In some alternatives, the gene deliverypolynucleotide is circular. In some alternatives, the gene deliverypolynucleotide is a minicircle. In some alternatives, the gene deliverypolynucleotide is at least 1 kB to 5 kB. In some alternatives, thepromoter region comprises an EF1 promoter sequence. In somealternatives, the fourth sequence comprises one, two, three, four, orfive genes that encode proteins. In some alternatives, the fourthsequence is codon optimized to reduce the total GC/AT ratio of thefourth sequence. In some alternatives, the fourth sequence is optimizedby codon optimization for expression in humans. In some alternatives,the fourth sequence is a consensus sequence generated from a pluralityof nucleic acids that encode a plurality of related proteins. In somealternatives, the fourth sequence is a consensus sequence generated froma plurality of nucleic acids that encode a plurality of relatedproteins, such as a plurality of antibody binding domains, which arespecific for the same epitope. In some alternatives, the plurality ofrelated proteins comprise a plurality of antibody binding domains,wherein the plurality of antibody binding domains are specific for thesame epitope. In some alternatives, the fifth sequence is codonoptimized to reduce the total GC/AT ratio of the fifth sequence. In somealternatives, the fifth sequence is optimized by codon optimization forexpression in humans. In some alternatives, the protein is a protein fortherapy. In some alternatives, the codon optimization and/or consensussequence is generated by comparing the variability of sequence and/ornucleobases utilized in a plurality of related sequences. In somealternatives, the protein comprises an antibody or a portion thereof,which may be humanized. In some alternatives, the double mutant ofdihydrofolate reductase comprises amino acid mutations of L22F and F31S.In some alternatives, the double mutant of dihydrofolate reductasecomprises amino acid mutations of L22F and F31S. In some alternatives,the introducing is performed by electroporation. In some alternatives,the selecting is performed by increasing selective pressure through theselective marker cassette. In some alternatives, the selection reagentcomprises an agent for selection. In some alternatives, the agent forselection is methotrexate. In some alternatives, the first concentrationrange is at least 50 nM-100 nM and the second concentration range is atleast 75 to 150 nM. In some alternatives, the first concentration is 50nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100 nM or any concentration that isbetween a range of concentrations defined by any two of theaforementioned concentrations, and the second concentration range is 75nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, or 150 nM orany concentration that is between a range of concentrations defined byany two of the aforementioned concentrations. In some alternatives, thefirst concentration range is at least 75 nM-150 nM and the secondconcentration range is at least 112.5 nM to 225 nM. In somealternatives, the first concentration is 75 nM, 85 nM, 95 nM, 105 nM,115 nM, 125 nM, 135 nM, 145 nM, or 150 nM or any concentration that isbetween a range of concentrations defined by any two of theaforementioned concentrations, and the second concentration range is 112nM, 122 nM, 132 nM, 142 nM, 152 nM, 162 nM, 172 nM, 182 nM, 192 nM, 202nM, 212 nM, or 225 nM or any concentration that is between a range ofconcentrations defined by any two of the aforementioned concentrations.In some alternatives, the first concentration range is at least 300nM-675 nM and the first concentration range is at least 450 nM to 1012nM. In some alternatives, the first concentration is 300 nM, 350 nM, 400nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, or 675 nM or anyconcentration that is between a range of concentrations defined by anytwo of the aforementioned concentrations, and the second concentrationrange is 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM,850 nM, 900 nM, 1000 nM, or 1012 nM or any concentration that is betweena range of concentrations defined by any two of the aforementionedconcentrations. In some alternatives, the first round of selectioncomprises exposing the T-cells to the selection agent for 2, 3, 4, 5, 6or 7 days before the second round of selection. In some alternatives,the second round of selection comprises exposing the T-cells to theselection agent for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or14 days or any time that is between a range of times defined by any twoof the aforementioned time points before isolation. In somealternatives, the subject is human.

Several of the material and methods are described in greater detailbelow.

Plasmids.

The pMC_T3/GFP-T2A-DHFRdm mini-circle (MC) plasmid that carries the T3SB transposon cassette containing an EF1a promoter, maxGFP gene,Thoseaasigna virus 2A peptide (T2A) and a double mutant of dihydrofolatereductase (DHFRdm) insensitive to methotrexate (MTX) was constructedusing pMC_T3/eGFP_IRES_FGFR (Nucleic Acids Research, 2012, 1-10doi:10.1093/nar/gks213, incorporated in its entirety herein) as abackbone, implementing the cloning strategy described previously (ColdSpring Harbor Protoc; 2012; doi:10.1101/pdb.ip067876) to create theGFP-T2A-DHFRdm cassette. MaxGFP (Lonza) andpEGFRt-T2A-IMPDHdm-T2A-DHFRdm (generously provided by Michael Jensen)plasmids were used as templates for PCR. BmtI and BamHI sites wereintroduced for swapping genes for fluorescent proteins. PlasmidMC_SB100X was described previously (Nucleic Acids Research, 2012, 1-10doi:10.1093/nar/gks213, incorporated in its entirety herein).Minicircles were produced and purified according to the SystemBiosciences user manual for minicircle DNA vector technology. Allplasmids were amplified under endotoxin free conditions using anEndofree Plasmid Kit (Qiagen).

H9 Culture and Transfection.

H9 cells were cultured in DMEM with 10% FBS. The optimized nucleofectionprotocol for H9 cells (Lonza) was followed (program X-001, NucleofectorKit V). Per nucleofection, 1×10⁶ cells were used with varying amounts ofMC DNA. Cells were grown for a week after nucleofection to achievestable transfection. For MTX selection, cells were cultured in DMEM with10% FBS supplemented with different concentrations of MTX.

Flow Cytometry Analysis.

Live cells were selected based on propidium iodide exclusion by addingpropidium iodide in the flow cytometry buffer to 2 ug/ml. Flow cytometryanalysis was carried out on a MACSQuant Analyzer (Miltenyi Biotec) andLSRII (BD Biosciences). Collected data was analyzed with FlowJosoftware. Appropriate negative controls (untransfected H9 cells with andwithout propidium iodide staining, as well as cells transfected withsingle genes for GFP, BFP, and mCherry) were used for compensation andgating. A Becton Dickinson FACSAria II was used for cell sorting. Partof flow cytometry work was conducted at the UW Immunology Flow CytometryFacility.

Determination of Transposon Copy Number.

Genomic DNA was extracted with Puregene Kit A according to themanufacturer's instructions (Qiagen), and qPCR was performed using a7300 Real-Time PCR System (Applied Biosystems) using Universal SYBRGreen Supermix (BioRad). Primers for qPCR were designed using Primer3software: maxGFP forward primer: 5′-ACAAGATCATCCGCAGCAAC-3′ (SEQ ID NO:4); reverse primer: 5′-TTGAAGTGCATGTGGCTGTC-3′(SEQ ID NO: 5); GAPDHforward primer: 5′-ACAACTTTGGTATCGTGGAAGG-3′(SEQ ID NO: 6); GAPDHreverse primer: 5′-GCCATCACGCCACAGTTTC-3′ (SEQ ID NO: 7). MaxGFP primersare specific for the maxGFP gene in the transposon. Standard curves weregenerated using genomic DNA of a H9 clone with a single insertion oftransposon (“gold standard”) obtained by limiting dilution method. Copynumber was calculated using the AACT method (Schmittgen, T. D. andLivak, K. J. (2008), incorporated in its entirety herein).

Characterization of SBTS Integration Distribution.

A population of T3/GFP-T2A-DHFRdm transfected-H9 cells selected with 200nM MTX was plated in 96 well plates at a concentration of 0.5 cells/wellin DMEM 10% FBS along with irradiated (5000 R) H9 feeder cells at 5,000cells/well. Plates were incubated for 2-3 weeks, after which clonalpopulations were moved to larger plates and expanded. GFP expression wasconfirmed by flow cytometry. Relative RT-qPCR analysis was performedusing DNA of 60 individual clones in order to determine transposon copynumber.

Optimization of Stable Gene Transfer to H9 Cells.

Minicircle constructs, which have bacterial plasmid sequences removed,were used for all gene transfer studies. Minicircles can be generated asdescribed previously by Kay et al. and colleagues (Chen, Z. Y.; He, C.Y.; Ehrhardt, A.; Kay, M. A. Molecular Therapy 2003, 8, 495-500 and Kay,M. A.; He, C. Y.; Chen, Z. Y. Nat. Biotechnol. 2010, 28, 1287-U96;incorporated herein by reference in their entirety). Three reporterminicircles containing transposons expressing different fluorescentproteins (maxGFP, mCherry, or BFP) under the EF1 alpha promoter wereconstructed. The selection gene, a double mutant of dihydrofolatereductase (DHFRdm) that confers metabolic resistance to MTX, was clonedin frame after the T2A sequence. The SB100X transposase gene was alsoprepared in a separate minicircle construct for co-delivery withtransposon minicircles.

There are four transposase binding sites in a transposon (two perinverted terminal repeat). Bound transposase were proposed to interactwith each other to promote juxtaposition of the two transposon ends.Overexpression of transposase has been hypothesized to lead toinhibition of transposition due to interaction of free transposase withbound transposase, thus preventing the juxtaposition step. Therefore,the optimal transposon/transposase ratio needed to be determined whetherthese genes are delivered on separate constructs. Reports of theinhibition phenomenon have been varied.

The efficiency of transient transfection were evaluated at 24 hourspost-nucleofection and at stable transposition (7 dayspost-nucleofection) at various transposon/transposase ratios using thereporter minicircle expressing maxGFP by flow cytometry. Attention isdrawn to FIG. 2, which shows the optimization of transposon:transposaseratio. The H9 T-cell line was used as the transfection test-bed. Initialtransfection efficiency ranged from 47.5%±2.2% to 66.9%±4.5%, increasingwith increased amount of transposase minicircle. In the absence oftransposase, minimal stable transfection (<1%) was detected 7 dayspost-nucleofection. The percentage of GFP⁺ cells increased with thetransposon/transposase ratio, reaching 39.2%±3.0% at 1:4 ratio, whichreflects 58.6% integration efficiency of the initialtransiently-integrated population. Higher ratios were not tested due toreduced cell viability. The overexpression inhibition effect was notobserved in this tested range of transposon/transposase ratios.Therefore, from the results, the transduction experiments were carriedout using this optimized transposon/transposase ratio of 1:4.

Selection of Engineered Cells with Methotrexate.

It was hypothesized that cells can be selected with multiple integrationevents using higher MTX concentrations due to increased selectionpressure for DHFRdm expression. Cells stably transduced with theT3/maxGFP-T2A-DHRFdm transposon were therefore grown in the presence ofincreasing MTX concentrations (ranging from 50 to 200 nM) and GFPexpression was evaluated by flow cytometry over 10 days. Attention isdrawn to FIG. 3, which shows the effect of methotrexate (MTX)concentration during selection. The initial selection efficiency,assessed with 3 days of MTX selection, was decreased with increasing MTXconcentration (FIG. 3, panel A). However, populations with >94% GFP+cells were obtained by 7 days post-selection under all conditions. Themean GFP fluorescence in GFP⁺ cells increased with selection pressure(FIG. 3, panel B); the mean fluorescence in cells selected with 200 nMMTX was 6.4-fold higher than unselected cells and 3.3-fold higher thancells selected with 50 nM MTX. As shown, the positive correlationbetween mean GFP expression in GFP+ cells and MTX concentration suggeststhat increasing MTX concentration selects for cells with increasedDHFRdm expression and therefore, multiple integration events.

The amplified cell populations selected with 2 weeks of MTX treatmentmaintained most of their transgene expression even upon MTX withdrawalup to 4 weeks. Attention is drawn to FIG. 4, which shows the transgenepersistence after methotrexate (MTX) withdrawal. Four weeks post-MTXwithdrawal, the GFP⁺ population remained >90% in all populations (FIG.4, panel A), although cells selected with 200 nM MTX had the highestGFP⁺ population (97%), likely due to selection of cells with multipleintegration events. The mean GFP expression in all populations decreasedby 21%, 27%, and 28% for 200 nM, 100 nM, and 50 nM MTX selection,respectively by 4 weeks post-MTX withdrawal (FIG. 4, panel B). As such,the decrease in mean GFP expression might be due to promoter silencingor preferential expansion of cells with lower GFP expression at theabsence of selective pressure.

Analysis of Distribution of Integration.

To test the hypothesis that increased MTX selection pressure wouldselect for cells with multiple integration events, the average number oftransposon copy numbers in MTX-selected cell populations was determinedusing RT-qPCR with GFP primers. First, a “gold standard” clone with asingle copy of integrated transposon was generated by limiting dilutionmethod. The average number of integrations in the originalstably-transduced population before MTX selection was determined byRT-qPCR analysis of the GFP⁺ cells obtained by cell sorting. A trend ofincreasing average transposon copy number with increasing selectionpressure was observed. Attention is drawn to FIG. 5A, which shows thetransposon copy number per human haploid genome. The average integrationevents in cells selected with 200 nM MTX was 2.1±0.45 compared to anaverage of 1.1±0.02 integration events in GFP+ cells before MTXselection. RT-qPCR was performed in triplicates and data represents asingle biological replica for the sorted population and 3 biologicalreplicas for MTX selection. Statistical difference was assessed byStudent's t-test.

The distribution of integration events in cells selected with 200 nM MTXwas then analyzed. Sixty clones were generated by limiting dilutionmethod, GFP expression confirmed by flow cytometry, genomic DNAisolated, and the number of GFP genes per haploid genome analyzed byRT-PCR. The distribution of integration events is shown in FIG. 5B. Mostclones (˜65%) contained multiple copies of GFP. The average number ofintegration events was 1.8 which correlates well with the averagetransposon copy number in the cell population selected with 200 nM MTX(FIG. 5A).

Demonstration of Multiplexed Gene Integration.

Since it was previously demonstrated that a majority of the populationof transduced cells amplified under 200 nM MTX selection pressurecontained multiple transposon copies, multiplexed gene integration wasthen assessed under these conditions. H9 cells were nucleofected withthree minicircles containing three different reporter genes (maxGFP,mCherry, and BFP) in transposon cassettes and the SB100X transposaseminicircle. Stably-transduced cells were then selected for 7 days with200 nM and cell population assessed by flow cytometry analysis.Attention is drawn to FIG. 6, which shows the flow cytometric analysisof H9 cell populations nucleofected with 3 minicircles carryingtransposons with different fluorescent proteins (MC_T3/GFP-T2A-DHFRdm,MC_T3/BFP-T2A-DHFRdm, MC_T3/mCherry-T2A-DHFRdm), 2 μg each and 6 μg ofMC_SB100X DNA at different time points after transfection. Initialtransfection efficiency assessed 24 hours after nucleofection, was 68%(FIG. 6 panel A). The stably transduced population was 37±1.4%,reflecting 54% integration efficiency. Of this population, 19±0.6%expressed two or three different fluorescent proteins. Stably-transducedcells grown for 1 week in the presence of 200 nM MTX were then analyzed;23±1.0% of this selected population expressed all three reporterproteins (FIG. 6 panel A). In order to further increase the populationof cells expressing triple transgenes, cells selected by 200 nM MTX weresubjected to a second selection step with increased MTX concentrations.Attention is drawn to FIG. 7, which shows a bar graph demonstrating theresults of an H9 cell population stably transfected with threetransposons selected with 200 nM MTX for a week and then exposed tohigher MTX concentrations of 500 and 1000 nM. As shown, cells that werecultured in 500 nM or 1000 nM MTX for an additional week resulting in anincreased population (38.5±1.0% and 53.1±0.3%, respectively) of cellsexpressing triple transgenes. Cell viability rebounded to ˜70% duringthe second round of selection due to further selection foroverexpression of the DHFRdm gene.

Stable Expression of Transposon DNA with Sleeping Beauty in T-Cells withMethotrexate Selection.

Freshly thawed peripheral blood mononuclear cells (PBMCs) wereelectroporated using Amaxa™ Nucleofector™ Technology. The cells weretransfected with 10 μg of minicircle GFP (MC_T3/GFP-T2A-DHFRdm) anddifferent amounts of SB100X hyperactive transposase (0, 5, or 10 μg).Control cells were transfected with either the non-minicircle pMAXGFPvector (10 ug) or with no DNA. The cells were then stimulated withMiltenyi Transact beads 4 to 6 hours after transfection in the presenceof IL-2 and IL-15. The cells were then aliquoted so that there were400,000 cells per well of a 96-well U-bottomed plate. The cells weretreated with methotrexate at 7 days after transfection with 0, 25, 50,or 100 nM of methotrexate. At days 2, 5, 7, 14, and 19, the cells werecounted by trypan blue, stained, and analyzed.

Attention is drawn to FIG. 8 which shows an example of the flow analysisof the lymphocytes expressing GFP after minicircle transfection. Singlecells (panel B) from the lymphocyte window (panel A) were analyzed forviability with the Invitrogen LIVE/DEAD red stain (panel C). Livelymphocytes were then analyzed for CD8 and GFP expression (panel D). Asshown in FIG. 8 panel D, after selecting with 50 nM methotrexate, themajority of lymphocytes were CD8+ and expressed GFP.

Stable Expression of Transposon DNA with Sleeping Beauty in T-Cellsafter One Week.

In order to assess the expression of the minicircle DNA in the weekbefore MTX selection, flow analysis was performed and then compared forcells transfected with pMAXGFP, 1:1 ratio of GFP transposon:SB100X, 1:2ratio of transposon:SB100X, mcGFP alone, or no DNA control. Attention isdrawn to FIG. 9, which shows the results of a FACS assay on cells at twodays (in the absence of Transact beads) and five days (in the presenceof Transact beads) after electroporation. As shown, there is a loss ofGFP expression over time without MTX. However, GFP expression persistsin cells transfected with GFP transposon DNA only if there wereco-transfected with SB100X transposase.

Attention is drawn to FIG. 10, which shows graphs of the levels of GFPexpression and cell growth from days 2 to 7. As shown in panel A of FIG.10, the amounts of percent GFP expression decreases over time (pMAXGFP(10 ug), mcGFP: MC_SB100X 1:1, and mcGFP: MC_SB100X 2:1). There was aslow increase of live cells in the presence of Transact beads (panel B),but not without the beads (panel C), indicating the importance of thebeads for cell growth.

Cell Selection with MTX for 7 Days and 12 Days.

After 1 week, samples of the transfected cells were exposed to differentlevels of MTX (25, 50, or 100 nM) to enrich for cells expressing theminicircle transposon. Cells that stably express the DHFRdmMTX-resistance gene as well as GFP due to transposase integration shouldsurvive higher MTX concentrations. Attention is drawn to FIG. 11, whichshows the results of a FACS assay of the transfected cells aftertreatment with 100 nM methotrexate for 7 days. In cells treated with 100nM MTX, only cells transfected with both transposon and transposase DNAexpress GFP. As shown, 100 nM MTX selection was effective with GFPexpression at both ratios of mcGFP to SB at a 2:1 mcGFP: MC_SB100X ratioand at a 1:1 mcGFP: MC_SB100X ratio after cell selection with MTX forseven days.

Attention is drawn to FIG. 12 and FIG. 13 which show the results of aFACS assay of the transfected cells after treatment with methotrexatefor 7 and 12 days, respectively. Scatter plots and CD8+/GFP expressionfor live lymphocytes are shown for each condition. Percent GFPexpression in lymphocytes is given in boxes in FIG. 12.

As shown in FIG. 12 at 7 days, cells treated with 0 or 25 nM MTX showabout 25% or 75% of the cells expressing GFP, respectively. In contrast,at least 90% of the cells express GFP at 50 and 100 nM MTX. As shown,MTX selection was equally effective for enrichment of GFP expression at50 nM and 100 nM, and at both ratios of mcGFP to SB at a 2:1 mcGFP:MC_SB100X ratio and a 1:1 mcGFP: MC_SB100X ratio. As expected, in theabsence of SB transposase and in the no DNA controls there is noappreciable GFP expression. Note that the expression of GFP is similarin CD8+ and CD8− lymphocytes.

Stable Expression of Transposon DNA with Sleeping Beauty in T-Cells withMethotrexate Selection-Cell Counts.

The cell growth of PBMC that stably expressing the transposon DNA underMTX selection, was later assessed. Note that due to stimulation withTransact beads and growth in the presence of IL2 and IL-15, the majorityof the surviving cells are T cells by 1 week. As shown in FIG. 14, theamounts of live cells following treatment with MTX at 0 nM, 25 nM, 50nM, and 100 nM methotrexate was determined with trypan blue cell countsat days 7, 14, and 19 days (days 0, 7, and 12 of MTX). In the control (0nM MTX), the number of live cells increased over time for all DNAconditions. However, in the presence of MTX, only the cells that weretransfected with both the SB transposase and the minicircle transposonthat coexpresses GFP and the DHFRdm resistance gene were able to divide,indicating that SB is required for stable expression of the transposon.

Stable Expression of Transposon DNA with Sleeping Beauty in T-Cells withMethotrexate Selection-GFP Expression.

The stable expression of transposon DNA with Sleeping Beauty in T-cellsduring MTX selection was assessed by determining the GFP expression ofthe transfected cells over 19 days. Attention is drawn to FIG. 14 whichshows increasing GFP expression over time in cells transfected withtransposon DNA and Sleeping Beauty in T-cells following methotrexateselection starting at day 7 (0, 25, 50, and 100 nM). As shown in thecontrol with no methotrexate selection from days 2, 5, 7, 14, and 19,the expression of GFP in the cells transfected with mcGFP and SB ismaintained at ˜20%, while the expression steadily decreases in the mcGFPalone and pMAXGFP controls. In the presence of MTX selection, GFPexpression increases over time, with highest levels seen with 50 and 100nM MTX. As shown, the ratios of mcGFP: MC_SB100X had no differencebetween a ratio of 1:1 and 2:1. Additionally there was minimaldifference in the mean fluorescence intensity in cells that were exposedto either 50 nM or 100 nM MTX. The low levels of GFP expression (˜20%)with mcGFP alone in the presence of MTX is likely due totransposon-independent stable integration, and the absolute number ofcells in these conditions is very low as shown in FIG. 14.

In one alternative, a gene delivery polynucleotide for stable insertionof a nucleic acid into a gene, wherein the nucleic acid for insertion isflanked by inverted terminal repeat gene sequences in the gene deliverypolynucleotide and wherein the gene delivery polynucleotide isselectable is provided, wherein the gene delivery polynucleotidecomprises a first sequence, wherein the first sequence comprises a firstinverted terminal repeat gene sequence, a second sequence, wherein thesecond sequence comprises a second inverted terminal repeat genesequence, a third sequence, wherein the third sequence comprises apromoter region sequence, a fourth sequence, wherein the fourth sequencecomprises at least one gene encoding a protein, and wherein the fourthsequence is optimized, a fifth sequence, wherein the fifth sequencecomprises at least one selectable marker cassette encoding a doublemutant of dihydrofolate reductase, wherein the double mutant ofdihydrofolate reductase has a 15,000 fold or about 15,000 fold reducedaffinity for methotrexate, wherein the methotrexate can be used as aselection mechanism to selectively amplify cells transduced with thegene delivery polynucleotide and wherein the fifth sequence isoptimized, a sixth sequence, wherein the sixth sequence comprises afirst attachment site (attP) and a seventh sequence, wherein the seventhsequence comprises a second attachment site (attB) wherein each of thefirst sequence, second sequence, third sequence, fourth sequence, fifthsequence, sixth sequence, and seventh sequence have a 5′ terminus and a3′ terminus, and wherein the 3′ terminus of the first sequencecomprising the first inverted terminal repeat gene sequence is adjacentto the 5′ terminus of the third sequence, the 3′ terminus of the thirdsequence is adjacent to the 5′ terminus of the fourth sequence, the 3′terminus of the fourth sequence is adjacent to the 5′ terminus of thefifth sequence and the 3′ terminus of the fifth sequence is adjacent tothe 5′ terminus of the second sequence comprising a second invertedterminal repeat. In some alternatives, the gene encoding the doublemutant of human dihydrofolate reductase comprises the DNA sequence:ATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACTTCCCCTGGCCACCGCTCAGGAATGAATCCAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAAGGAGCTCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAGAATGATTAA (SEQ ID NO: 2). In some alternatives, the doublemutant of human dihydrofolate reductase comprises the protein sequence:MVGSLNCIVA VSQNMGIGKN GDFPWPPLRN ESRYFQRMTT TSSVEGKQNL VIMGKKTWFSIPEKNRPLKG RINLVLSREL KEPPQGAHFL SRSLDDALKL TEQPELANKV DMVWIVGGSSVYKEAMNHPG HLKLFVTRIM QDFESDTFFP EIDLEKYKLL PEYPGVLSDV QEEKGIKYKFEVYEKND (SEQ ID NO: 3). In some alternatives, the gene deliverypolynucleotide is circular. In some alternatives, the gene deliverypolynucleotide is at least 1 kB to 5 kB. In some alternatives, the genedelivery polynucleotide is a minicircle. In some alternatives, the genedelivery polynucleotide is a minicircle. In some alternatives, thepromoter region comprises an EF1 promoter sequence. In somealternatives, the fourth sequence comprises one, two, three, four, orfive genes that encode proteins. In some alternatives, the fourthsequence is codon optimized to reduce the total GC/AT ratio of thefourth sequence. In some alternatives, the fourth sequence is optimizedby codon optimization for expression in humans. In some alternatives,the fourth sequence is a consensus sequence generated from a pluralityof nucleic acids that encode a plurality of related proteins. In somealternatives, the fourth sequence is a consensus sequence generated froma plurality of nucleic acids that encode a plurality of relatedproteins, such as a plurality of antibody binding domains, which arespecific for the same epitope. In some alternatives, the plurality ofrelated proteins comprise a plurality of antibody binding domains,wherein the plurality of antibody binding domains are specific for thesame epitope. In some alternatives, the fifth sequence is codonoptimized to reduce the total GC/AT ratio of the fifth sequence. In somealternatives, the fifth sequence is optimized by codon optimization forexpression in humans. In some alternatives, the protein is a protein fortherapy. In some alternatives, the codon optimization and/or consensussequence is generated by comparing the variability of sequence and/ornucleobases utilized in a plurality of related sequences. In somealternatives, the protein comprises an antibody or a portion thereof,which may be humanized. In some alternatives, the double mutant ofdihydrofolate reductase comprises amino acid mutations of L22F and F31S.

In some alternatives, a method of generating engineered multiplexedT-cells for adoptive T-cell immunotherapy is provided, wherein themethod comprises providing a gene delivery polynucleotide as describedherein, introducing the gene delivery polynucleotide into a T-cell,providing a vector encoding a Sleeping Beauty transposase, introducingthe vector encoding the Sleeping Beauty transposase into the T-cell,selecting the cells comprising the gene delivery polynucleotide whereinselecting comprises a first round of selection and a second round ofselection, wherein the first round of selection comprises adding aselection reagent at a first concentration range and the second round ofselection comprises adding the selection reagent at a secondconcentration range, wherein the second concentration range is higherthan the first concentration range and, wherein the second concentrationrange is at least 1.5 fold higher than that of the first concentrationrange and isolating the T-cells expressing a phenotype under selectivepressure. In some alternatives, the gene delivery polynucleotidecomprises a first sequence, wherein the first sequence comprises a firstinverted terminal repeat gene sequence, a second sequence, wherein thesecond sequence comprises a second inverted terminal repeat genesequence, a third sequence, wherein the third sequence comprises apromoter region sequence, a fourth sequence, wherein the fourth sequencecomprises at least one gene encoding a protein, and wherein the fourthsequence is optimized, a fifth sequence, wherein the fifth sequencecomprises at least one selectable marker cassette encoding a doublemutant of dihydrofolate reductase, wherein the double mutant ofdihydrofolate reductase has a 15,000 fold or about 15,000 fold reducedaffinity for methotrexate, wherein the methotrexate can be used as aselection mechanism to selectively amplify cells transduced with thegene delivery polynucleotide and wherein the fifth sequence isoptimized, a sixth sequence, wherein the sixth sequence comprises afirst attachment site (attP) and a seventh sequence, wherein the seventhsequence comprises a second attachment site (attB) wherein each of thefirst sequence, second sequence, third sequence, fourth sequence, fifthsequence, sixth sequence, and seventh sequence have a 5′ terminus and a3′ terminus, and wherein the 3′ terminus of the first sequencecomprising the first inverted terminal repeat gene sequence is adjacentto the 5′ terminus of the third sequence, the 3′ terminus of the thirdsequence is adjacent to the 5′ terminus of the fourth sequence, the 3′terminus of the fourth sequence is adjacent to the 5′ terminus of thefifth sequence and the 3′ terminus of the fifth sequence is adjacent tothe 5′ terminus of the second sequence comprising a second invertedterminal repeat. In some alternatives, the gene encoding the doublemutant of human dihydrofolate reductase comprises the DNA sequence:ATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACTTCCCCTGGCCACCGCTCAGGAATGAATCCAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAAGGAGCTCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAGAATGATTAA (SEQ ID NO: 2). In some alternatives, the doublemutant of human dihydrofolate reductase comprises the protein sequence:MVGSLNCIVA VSQNMGIGKN GDFPWPPLRN ESRYFQRMTT TSSVEGKQNL VIMGKKTWFSIPEKNRPLKG RINLVLSREL KEPPQGAHFL SRSLDDALKL TEQPELANKV DMVWIVGGSSVYKEAMNHPG HLKLFVTRIM QDFESDTFFP EIDLEKYKLL PEYPGVLSDV QEEKGIKYKFEVYEKND (SEQ ID NO: 3). In some alternatives, the gene deliverypolynucleotide is circular. In some alternatives, the gene deliverypolynucleotide is at least 1 kB to 5 kB. In some alternatives, the genedelivery polynucleotide is a minicircle. In some alternatives, thepromoter region comprises an EF1 promoter sequence. In somealternatives, the fourth sequence comprises one, two, three, four, orfive genes that encode proteins. In some alternatives, the fourthsequence is codon optimized to reduce the total GC/AT ratio of thefourth sequence. In some alternatives, the fourth sequence is optimizedby codon optimization for expression in humans. In some alternatives,the fourth sequence is a consensus sequence generated from a pluralityof nucleic acids that encode a plurality of related proteins. In somealternatives, the fourth sequence is a consensus sequence generated froma plurality of nucleic acids that encode a plurality of relatedproteins, such as a plurality of antibody binding domains, which arespecific for the same epitope. In some alternatives, the plurality ofrelated proteins comprise a plurality of antibody binding domains,wherein the plurality of antibody binding domains are specific for thesame epitope. In some alternatives, the fifth sequence is codonoptimized to reduce the total GC/AT ratio of the fifth sequence. In somealternatives, the fifth sequence is optimized by codon optimization forexpression in humans. In some alternatives, the protein is a protein fortherapy. In some alternatives, the codon optimization and/or consensussequence is generated by comparing the variability of sequence and/ornucleobases utilized in a plurality of related sequences. In somealternatives, the protein comprises an antibody or a portion thereof,which may be humanized. In some alternatives, the double mutant ofdihydrofolate reductase comprises amino acid mutations of L22F and F31S.In some alternatives, the introducing is performed by electroporation.In some alternatives, the selecting is performed by increasing selectivepressure through the selective marker cassette. In some alternatives,the selection reagent comprises an agent for selection. In somealternatives, the agent for selection is methotrexate. In somealternatives, the first concentration range is at least 50 nM-100 nM andthe second concentration range is at least 75 to 150 nM. In somealternatives, the first concentration is 50 nM, 60 nM, 70 nM, 80 nM, 90nM, or 100 nM or any concentration that is between a range ofconcentrations defined by any two of the aforementioned concentrations,and the second concentration range is 75 nM, 80 nM, 90 nM, 100 nM, 110nM, 120 nM, 130 nM, 140 nM, or 150 nM or any concentration that isbetween a range of concentrations defined by any two of theaforementioned concentrations. In some alternatives, the firstconcentration range is at least 75 nM-150 nM and the secondconcentration range is at least 112.5 nM to 225 nM. In somealternatives, the first concentration is 75 nM, 85 nM, 95 nM, 105 nM,115 nM, 125 nM, 135 nM, 145 nM, or 150 nM or any concentration that isbetween a range of concentrations defined by any two of theaforementioned concentrations, and the second concentration range is 112nM, 122 nM, 132 nM, 142 nM, 152 nM, 162 nM, 172 nM, 182 nM, 192 nM, 202nM, 212 nM, or 225 nM or any concentration that is between a range ofconcentrations defined by any two of the aforementioned concentrations.In some alternatives, the first concentration range is at least 300nM-675 nM and the first concentration range is at least 450 nM to 1012nM. In some alternatives, the first concentration is 300 nM, 350 nM, 400nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, or 675 nM or anyconcentration that is between a range of concentrations defined by anytwo of the aforementioned concentrations, and the second concentrationrange is 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM,850 nM, 900 nM, 1000 nM, or 1012 nM or any concentration that is betweena range of concentrations defined by any two of the aforementionedconcentrations. In some alternatives, the first round of selectioncomprises exposing the T-cells to the selection agent for 2, 3, 4, 5, 6or 7 days before the second round of selection. In some alternatives,the second round of selection comprises exposing the T-cells to theselection agent for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or14 days or any time that is between a range of times defined by any twoof the aforementioned time points before isolation.

In some alternatives, a method of increasing protein production in aT-cell is provided, wherein the method comprises providing apolynucleotide described herein, introducing the polynucleotide into acell, providing a vector encoding a Sleeping Beauty transposase,introducing the vector encoding the Sleeping Beauty transposase into theT-cell, selecting the cells comprising the gene delivery polynucleotidewherein selecting comprises a first round of selection and a secondround of selection, wherein the first round of selection comprisesadding a selection reagent at a first concentration range and the secondround of selection comprises adding the selection reagent at a secondconcentration range, wherein the second concentration range is higherthan the first concentration range and, wherein the second concentrationrange is at least 1.5 fold higher than that of the first concentrationrange and isolating the cells expressing a phenotype under selectivepressure. In some alternatives, the introducing is performed byelectroporation. In some alternatives, the selecting is performed byincreasing selective pressure through the selective marker cassette. Insome alternatives, the selection reagent comprises an agent forselection. In some alternatives, the agent for selection ismethotrexate. In some alternatives, the first concentration range is atleast 50 nM-100 nM and the second concentration range is at least 75 to150 nM. In some alternatives, the first concentration is 50 nM, 60 nM,70 nM, 80 nM, 90 nM, or 100 nM or any concentration that is between arange of concentrations defined by any two of the aforementionedconcentrations, and the second concentration range is 75 nM, 80 nM, 90nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, or 150 nM or anyconcentration that is between a range of concentrations defined by anytwo of the aforementioned concentrations. In some alternatives, thefirst concentration range is at least 75 nM-150 nM and the secondconcentration range is at least 112.5 nM to 225 nM. In somealternatives, the first concentration is 75 nM, 85 nM, 95 nM, 105 nM,115 nM, 125 nM, 135 nM, 145 nM, or 150 nM or any concentration that isbetween a range of concentrations defined by any two of theaforementioned concentrations, and the second concentration range is 112nM, 122 nM, 132 nM, 142 nM, 152 nM, 162 nM, 172 nM, 182 nM, 192 nM, 202nM, 212 nM, or 225 nM or any concentration that is between a range ofconcentrations defined by any two of the aforementioned concentrations.In some alternatives, the first concentration range is at least 300nM-675 nM and the first concentration range is at least 450 nM to 1012nM. In some alternatives, the first concentration is 300 nM, 350 nM, 400nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, or 675 nM or anyconcentration that is between a range of concentrations defined by anytwo of the aforementioned concentrations, and the second concentrationrange is 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM,850 nM, 900 nM, 1000 nM, or 1012 nM or any concentration that is betweena range of concentrations defined by any two of the aforementionedconcentrations. In some alternatives, the first round of selectioncomprises exposing the T-cells to the selection agent for 2, 3, 4, 5, 6or 7 days before the second round of selection. In some alternatives,the second round of selection comprises exposing the T-cells to theselection agent for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or14 days or any time that is between a range of times defined by any twoof the aforementioned time points before isolation.

In some alternatives, the gene delivery polynucleotide comprises a firstsequence, wherein the first sequence comprises a first inverted terminalrepeat gene sequence, a second sequence, wherein the second sequencecomprises a second inverted terminal repeat gene sequence, a thirdsequence, wherein the third sequence comprises a promoter regionsequence, a fourth sequence, wherein the fourth sequence comprises atleast one gene encoding a protein, and wherein the fourth sequence isoptimized, a fifth sequence, wherein the fifth sequence comprises atleast one selectable marker cassette encoding a double mutant ofdihydrofolate reductase, wherein the double mutant of dihydrofolatereductase has a 15,000 fold or about 15,000 fold reduced affinity formethotrexate, wherein the methotrexate can be used as a selectionmechanism to selectively amplify cells transduced with the gene deliverypolynucleotide and wherein the fifth sequence is optimized, a sixthsequence, wherein the sixth sequence comprises a first attachment site(attP) and a seventh sequence, wherein the seventh sequence comprises asecond attachment site (attB) wherein each of the first sequence, secondsequence, third sequence, fourth sequence, fifth sequence, sixthsequence, and seventh sequence have a 5′ terminus and a 3′ terminus, andwherein the 3′ terminus of the first sequence comprising the firstinverted terminal repeat gene sequence is adjacent to the 5′ terminus ofthe third sequence, the 3′ terminus of the third sequence is adjacent tothe 5′ terminus of the fourth sequence, the 3′ terminus of the fourthsequence is adjacent to the 5′ terminus of the fifth sequence and the 3′terminus of the fifth sequence is adjacent to the 5′ terminus of thesecond sequence comprising a second inverted terminal repeat. In somealternatives, the gene encoding the double mutant of human dihydrofolatereductase comprises the DNA sequence:ATGGTTGGTTCGCTAAACTGCATCGTCGCTGTGTCCCAGAACATGGGCATCGGCAAGAACGGGGACTTCCCCTGGCCACCGCTCAGGAATGAATCCAGATATTTCCAGAGAATGACCACAACCTCTTCAGTAGAAGGTAAACAGAATCTGGTGATTATGGGTAAGAAGACCTGGTTCTCCATTCCTGAGAAGAATCGACCTTTAAAGGGTAGAATTAATTTAGTTCTCAGCAGAGAACTCAAGGAACCTCCACAAGGAGCTCATTTTCTTTCCAGAAGTCTAGATGATGCCTTAAAACTTACTGAACAACCAGAATTAGCAAATAAAGTAGACATGGTCTGGATAGTTGGTGGCAGTTCTGTTTATAAGGAAGCCATGAATCACCCAGGCCATCTTAAACTATTTGTGACAAGGATCATGCAAGACTTTGAAAGTGACACGTTTTTTCCAGAAATTGATTTGGAGAAATATAAACTTCTGCCAGAATACCCAGGTGTTCTCTCTGATGTCCAGGAGGAGAAAGGCATTAAGTACAAATTTGAAGTATATGAGAAGAATGATTAA (SEQ ID NO: 2). In some alternatives, the doublemutant of human dihydrofolate reductase comprises the protein sequence:MVGSLNCIVA VSQNMGIGKN GDFPWPPLRN ESRYFQRMTT TSSVEGKQNL VIMGKKTWFSIPEKNRPLKG RINLVLSREL KEPPQGAHFL SRSLDDALKL TEQPELANKV DMVWIVGGSSVYKEAMNHPG HLKLFVTRIM QDFESDTFFP EIDLEKYKLL PEYPGVLSDV QEEKGIKYKFEVYEKND (SEQ ID NO: 3). In some alternatives, the gene deliverypolynucleotide is circular. In some alternatives, the gene deliverypolynucleotide is at least 1 kB to 5 kB. In some alternatives, the genedelivery polynucleotide is a minicircle. In some alternatives, the genedelivery polynucleotide is a minicircle. In some alternatives, thepromoter region comprises an EF1 promoter sequence. In somealternatives, the fourth sequence comprises one, two, three, four, orfive genes that encode proteins. In some alternatives, the fourthsequence is codon optimized to reduce the total GC/AT ratio of thefourth sequence. In some alternatives, the fourth sequence is optimizedby codon optimization for expression in humans. In some alternatives,the fourth sequence is a consensus sequence generated from a pluralityof nucleic acids that encode a plurality of related proteins. In somealternatives, the fourth sequence is a consensus sequence generated froma plurality of nucleic acids that encode a plurality of relatedproteins, such as a plurality of antibody binding domains, which arespecific for the same epitope. In some alternatives, the plurality ofrelated proteins comprise a plurality of antibody binding domains,wherein the plurality of antibody binding domains are specific for thesame epitope. In some alternatives, the fifth sequence is codonoptimized to reduce the total GC/AT ratio of the fifth sequence. In somealternatives, the fifth sequence is optimized by codon optimization forexpression in humans. In some alternatives, the codon optimizationand/or consensus sequence is generated by comparing the variability ofsequence and/or nucleobases utilized in a plurality of relatedsequences. In some alternatives, the protein comprises an antibody or aportion thereof, which may be humanized. In some alternatives, thedouble mutant of dihydrofolate reductase comprises amino acid mutationsof L22F and F31S. In some alternatives, the introducing is performed byelectroporation. In some alternatives, the selecting is performed byincreasing selective pressure through the selective marker cassette. Insome alternatives, the selection reagent comprises an agent forselection. In some alternatives, the agent for selection ismethotrexate. In some alternatives, the first concentration range is atleast 50 nM-100 nM and the second concentration range is at least 75 to150 nM. In some alternatives, the first concentration is 50 nM, 60 nM,70 nM, 80 nM, 90 nM, or 100 nM or any concentration that is between arange of concentrations defined by any two of the aforementionedconcentrations, and the second concentration range is 75 nM, 80 nM, 90nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, or 150 nM or anyconcentration that is between a range of concentrations defined by anytwo of the aforementioned concentrations. In some alternatives, thefirst concentration range is at least 75 nM-150 nM and the secondconcentration range is at least 112.5 nM to 225 nM. In somealternatives, the first concentration is 75 nM, 85 nM, 95 nM, 105 nM,115 nM, 125 nM, 135 nM, 145 nM, or 150 nM or any concentration that isbetween a range of concentrations defined by any two of theaforementioned concentrations, and the second concentration range is 112nM, 122 nM, 132 nM, 142 nM, 152 nM, 162 nM, 172 nM, 182 nM, 192 nM, 202nM, 212 nM, or 225 nM or any concentration that is between a range ofconcentrations defined by any two of the aforementioned concentrations.In some alternatives, the first concentration range is at least 300nM-675 nM and the first concentration range is at least 450 nM to 1012nM. In some alternatives, the first concentration is 300 nM, 350 nM, 400nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, or 675 nM or anyconcentration that is between a range of concentrations defined by anytwo of the aforementioned concentrations, and the second concentrationrange is 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM,850 nM, 900 nM, 1000 nM, or 1012 nM or any concentration that is betweena range of concentrations defined by any two of the aforementionedconcentrations. In some alternatives, the first round of selectioncomprises exposing the T-cells to the selection agent for 2, 3, 4, 5, 6or 7 days before the second round of selection. In some alternatives,the second round of selection comprises exposing the T-cells to theselection agent for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or14 days or any time that is between a range of times defined by any twoof the aforementioned time points before isolation.

In some alternatives, a method of treating, inhibiting, or amelioratingcancer or a disease in a subject, the method comprising administering tothe subject a modified T-cell as described herein. In some alternatives,the subject is human.

With respect to the use of substantially any plural and/or singularterms herein, those having skill in the art can translate from theplural to the singular and/or from the singular to the plural as isappropriate to the context and/or application. The varioussingular/plural permutations may be expressly set forth herein for sakeof clarity.

It will be understood by those within the art that, in general, termsused herein, and especially in the appended claims (e.g., bodies of theappended claims) are generally intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.). It will be further understood by those within the art that if aspecific number of an introduced claim recitation is intended, such anintent will be explicitly recited in the claim, and in the absence ofsuch recitation no such intent is present. For example, as an aid tounderstanding, the following appended claims may contain usage of theintroductory phrases “at least one” and “one or more” to introduce claimrecitations. However, the use of such phrases should not be construed toimply that the introduction of a claim recitation by the indefinitearticles “a” or “an” limits any particular claim containing suchintroduced claim recitation to embodiments containing only one suchrecitation, even when the same claim includes the introductory phrases“one or more” or “at least one” and indefinite articles such as “a” or“an” (e.g., “a” and/or “an” should be interpreted to mean “at least one”or “one or more”); the same holds true for the use of definite articlesused to introduce claim recitations. In addition, even if a specificnumber of an introduced claim recitation is explicitly recited, thoseskilled in the art will recognize that such recitation should beinterpreted to mean at least the recited number (e.g., the barerecitation of “two recitations,” without other modifiers, means at leasttwo recitations, or two or more recitations). Furthermore, in thoseinstances where a convention analogous to “at least one of A, B, and C,etc.” is used, in general such a construction is intended in the senseone having skill in the art would understand the convention (e.g., “asystem having at least one of A, B, and C” would include but not belimited to systems that have A alone, B alone, C alone, A and Btogether, A and C together, B and C together, and/or A, B, and Ctogether, etc.). In those instances where a convention analogous to “atleast one of A, B, or C, etc.” is used, in general such a constructionis intended in the sense one having skill in the art would understandthe convention (e.g., “a system having at least one of A, B, or C” wouldinclude but not be limited to systems that have A alone, B alone, Calone, A and B together, A and C together, B and C together, and/or A,B, and C together, etc.). It will be further understood by those withinthe art that virtually any disjunctive word and/or phrase presenting twoor more alternative terms, whether in the description, claims, ordrawings, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms. Forexample, the phrase “A or B” will be understood to include thepossibilities of “A” or “B” or “A and B.”

In addition, where features or aspects of the disclosure are describedin terms of Markush groups, those skilled in the art will recognize thatthe disclosure is also thereby described in terms of any individualmember or subgroup of members of the Markush group

1. A gene delivery polynucleotide for stable insertion of a nucleic acid into an oligonucleotide, wherein the nucleic acid for insertion is flanked by inverted terminal repeat gene sequences in the gene delivery polynucleotide and, wherein the gene delivery polynucleotide is selectable, the gene delivery polynucleotide comprising: a first sequence, wherein the first sequence comprises a first inverted terminal repeat gene sequence; a second sequence, wherein the second sequence comprises a second inverted terminal repeat gene sequence; a third sequence, wherein the third sequence comprises a promoter region sequence; a fourth sequence, wherein the fourth sequence comprises at least one gene, wherein the at least one gene encodes a protein or encodes a sequence for mRNA transcription, and wherein the fourth sequence is optimized; a fifth sequence, wherein the fifth sequence comprises at least one selectable marker cassette encoding a double mutant of dihydrofolate reductase, wherein the double mutant of dihydrofolate reductase has a 15,000 fold or about 15,000 fold reduced affinity for methotrexate, wherein the methotrexate can be used to select for cells transduced with the gene delivery polynucleotide to enhance the ratio of cells expressing the at least one gene and wherein the fifth sequence is optimized; a sixth sequence, wherein the sixth sequence comprises a first attachment site (attP); and a seventh sequence, wherein the seventh sequence comprises a second attachment site (attB); wherein each of the first sequence, second sequence, third sequence, fourth sequence, fifth sequence, sixth sequence, and seventh sequence have a 5′ terminus and a 3′ terminus, and wherein the 3′ terminus of the first sequence comprising the first inverted terminal repeat gene sequence is adjacent to the 5′ terminus of the third sequence, the 3′ terminus of the third sequence is adjacent to the 5′ terminus of the fourth sequence, the 3′ terminus of the fourth sequence is adjacent to the 5′ terminus of the fifth sequence and the 3′ terminus of the fifth sequence is adjacent to the 5′ terminus of the second sequence comprising a second inverted terminal repeat. 2.-64. (canceled) 